Immunostimulatory sequence oligonucleotides and methods of using the same

ABSTRACT

The invention provides immunomodulatory polynucleotides and methods for immunomodulation of individuals using the immunomodulatory polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/741,720, filed on Dec. 18, 2003, now U.S. Pat. No. 7,745,606, whichclaims priority to (1) U.S. Provisional Patent Application No.60/436,122, filed on Dec. 23, 2002; (2) U.S. Provisional PatentApplication No. 60/447,885, filed on Feb. 13, 2003; and (3) U.S.Provisional Patent Application No. 60/467,546, filed on May 1, 2003, thedisclosures of which are herein incorporated by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to immunomodulatory polynucleotides. Italso relates to the administration of the polynucleotides to modulate animmune response.

BACKGROUND OF THE INVENTION

The type of immune response generated to infection or other antigenicchallenge can generally be distinguished by the subset of T helper (Th)cells involved in the response. The Th1 subset is responsible forclassical cell-mediated functions such as delayed-type hypersensitivityand activation of cytotoxic T lymphocytes (CTLs), whereas the Th2 subsetfunctions more effectively as a helper for B-cell activation. The typeof immune response to an antigen is generally influenced by thecytokines produced by the cells responding to the antigen. Differencesin the cytokines secreted by Th1 and Th2 cells are believed to reflectdifferent biological functions of these two subsets. See, for example,Romagnani (2000) Ann. Allergy Asthma Immunol. 85:9-18.

The Th1 subset may be particularly suited to respond to viralinfections, intracellular pathogens, and tumor cells because it secretesIL-2 and IFN-γ, which activate CTLs. The Th2 subset may be more suitedto respond to free-living bacteria and helminthic parasites and maymediate allergic reactions, since IL-4 and IL-5 are known to induce IgEproduction and eosinophil activation, respectively. In general, Th1 andTh2 cells secrete distinct patterns of cytokines and so one type ofresponse can moderate the activity of the other type of response. Ashift in the Th1/Th2 balance can result in an allergic response, forexample, or, alternatively, in an increased CTL response.

For many infectious diseases, such as tuberculosis and malaria, Th2-typeresponses are of little protective value against infection. Proposedvaccines using small peptides derived from the target antigen and othercurrently used antigenic agents that avoid use of potentially infectiveintact viral particles, do not always elicit the immune responsenecessary to achieve a therapeutic effect. Protein-based vaccinestypically induce Th2-type immune responses, characterized by high titersof neutralizing antibodies but without significant cell-mediatedimmunity.

Moreover, some types of antibody responses are inappropriate in certainindications, most notably in allergy where an IgE antibody response canresult in anaphylactic shock. Generally, allergic responses also involveTh2-type immune responses. Allergic responses, including those ofallergic asthma, are characterized by an early phase response, whichoccurs within seconds to minutes of allergen exposure and ischaracterized by cellular degranulation, and a late phase response,which occurs 4 to 24 hours later and is characterized by infiltration ofeosinophils into the site of allergen exposure. Specifically, during theearly phase of the allergic response, allergen cross-links IgEantibodies on basophils and mast cells, which in turn triggersdegranulation and the subsequent release of histamine and othermediators of inflammation from mast cells and basophils. During the latephase response, eosinophils infiltrate into the site of allergenexposure (where tissue damage and dysfunction result).

Antigen immunotherapy for allergic disorders involves the subcutaneousinjection of small, but gradually increasing amounts, of antigen. Suchimmunization treatments present the risk of inducing IgE-mediatedanaphylaxis and do not efficiently address the cytokine-mediated eventsof the allergic late phase response. Thus far, this approach has yieldedonly limited success.

Administration of certain DNA sequences, generally known asimmunostimulatory sequences, induces an immune response with a Th1-typebias as indicated by secretion of Th1-associated cytokines.Administration of an immunostimulatory polynucleotide with an antigenresults in a Th1-type immune response to the administered antigen. Romanet al. (1997) Nature Med. 3:849-854. For example, mice injectedintradermally with Escherichia coli (E. coli) β-galactosidase (β-Gal) insaline or in the adjuvant alum responded by producing specific IgG1 andIgE antibodies, and CD4⁺ cells that secreted IL-4 and IL-5, but notIFN-γ, demonstrating that the T cells were predominantly of the Th2subset. However, mice injected intradermally (or with a tyne skinscratch applicator) with plasmid DNA (in saline) encoding β-Gal andcontaining an immunostimulatory sequence responded by producing IgG2aantibodies and CD4⁺ cells that secreted IFN-γ, but not IL-4 and IL-5,demonstrating that the T cells were predominantly of the Th1 subset.Moreover, specific IgE production by the plasmid DNA-injected mice wasreduced 66-75%. Raz et al. (1996) Proc. Natl. Acad. Sci. USA93:5141-5145. In general, the response to naked DNA immunization ischaracterized by production of IL-2, TNFα and IFN-γ byantigen-stimulated CD4⁺ T cells, which is indicative of a Th1-typeresponse. This is particularly important in treatment of allergy andasthma as shown by the decreased IgE production. The ability ofimmunostimulatory polynucleotides to stimulate a Th1-type immuneresponse has been demonstrated with bacterial antigens, viral antigensand with allergens (see, for example, WO 98/55495).

References describing immunostimulatory activity of polynucleotidesinclude: Krug et al. (2001) Eur. J. Immunol. 31:3026; Bauer et al.(2001) J. Immunol. 166:5000; Klinman et al. (1999) Vaccine 17:19;Jahn-Schmid et al. (1999) J. Allergy Clin. Immunol. 104:1015; Tighe etal. (2000) Eur. J. Immunol. 30:1939; Shirota et al. (2000) J. Immunol.164:5575; Klinman et al. (1999) Infect. Immun. 67:5658; Sur et al.(1999) J. Immunol. 162:6284; Magone et al. (2000) Eur. J. Immunol.30:1841; Kawarada et al. (2001) J. Immunol. 167:5247; Kranzer et al.(2000) Immunology 99:170; Krug et al. (2001) Eur. J. Immunol. 31:2154;Hartmann et al. (2000) J. Immunol. 164:944; Bauer et al. (1999)Immunology 97:699; Fujieda et al. (2000) Am. J. Respir. Crit. Care Med.162:232; Krieg (2002) Annu. Rev. Immunol. 20:709; Verthelyi et al.(2002) J. Immunol. 168:1659; Hornung et al. (2002) J. Immunol. 168:4531;Yamamoto et al. (2000) Springer Semin. Immunopathol. 22:35; Lee et al.(2000) J. Immunol. 165:3631; Gursel et al. (2002) J. Leukoc. Biol.71:813; Gursel et al. (2002) Eur. J. Immunol. 32:2617; Broide et al.(2001) J. Clin. Immunol. 21:175; Zhu et al. (2001) Immunology 103:226;Klinman et al. (2002)Microbes Infect. 4:897; Hartmann et al. (2000) J.Immunol. 164:1617; Krieg (1999) Biochim. Biophys. Acta 1489:107; Dalpkeet al. (2002) Immunology 106:102; Yu et al. (2002) Biochem. Biophys.Res. Commun. 297:83; Hafner et al. (2001) Cancer Res. 61:5523; Zwavelinget al. (2002) J. Immunol. 169:350; Davis et al. (2000) Vaccine 18:1920;Gierynska et al. (2002) J. Virol. 76:6568; Lipford et al. (2000) J.Immunol. 165:1228; Freidag et al. (2000) Infect. Immun. 68:2948;Dieudonne et al. (2001) J. Allergy Clin. Immunol. 107:S233.

Other references describing immunostimulatory sequences include: Krieget al. (1989) J. Immunol. 143:2448-2451; Tokunaga et al. (1992)Microbiol. Immunol. 36:55-66; Kataoka et al. (1992) Jpn. J. Cancer Res.83:244-247; Yamamoto et al. (1992) J. Immunol. 148:4072-4076; Mojcik etal. (1993) Clin. Immuno. and Immunopathol. 67:130-136; Branda et al.(1993) Biochem. Pharmacol. 45:2037-2043; Pisetsky et al. (1994) LifeSci. 54(2):101-107; Yamamoto et al. (1994a) Antisense Research andDevelopment. 4:119-122; Yamamoto et al. (1994b) Jpn. J. Cancer Res.85:775-779; Raz et al. (1994) Proc. Natl. Acad. Sci. USA 91:9519-9523;Kimura et al. (1994) J. Biochem. (Tokyo) 116:991-994; Krieg et al.(1995) Nature 374:546-549; Pisetsky et al. (1995) Ann. N.Y. Acad. Sci.772:152-163; Pisetsky (1996a) J. Immunol. 156:421-423; Pisetsky (1996b)Immunity 5:303-310; Zhao et al. (1996) Biochem. Pharmacol. 51:173-182;Yi et al. (1996) J. Immunol. 156:558-564; Krieg (1996) Trends Microbiol.4(2):73-76; Krieg et al. (1996) Antisense Nucleic Acid Drug Dev.6:133-139; Klinman et al. (1996) Proc. Natl. Acad. Sci. USA.93:2879-2883; Raz et al. (1996); Sato et al. (1996) Science 273:352-354;Stacey et al. (1996) J. Immunol. 157:2116-2122; Ballas et al. (1996) J.Immunol. 157:1840-1845; Branda et al. (1996) J. Lab. Clin. Med.128:329-338; Sonehara et al. (1996) J. Interferon and Cytokine Res.16:799-803; Klinman et al. (1997) J. Immunol. 158:3635-3639; Sparwasseret al. (1997) Eur. J. Immunol. 27:1671-1679; Roman et al. (1997); Carsonet al. (1997) J. Exp. Med. 186:1621-1622; Chace et al. (1997) Clin.Immunol. and Immunopathol. 84:185-193; Chu et al. (1997) J. Exp. Med.186:1623-1631; Lipford et al. (1997a) Eur. J. Immunol. 27:2340-2344;Lipford et al. (1997b) Eur. J. Immunol. 27:3420-3426; Weiner et al.(1997) Proc. Natl. Acad. Sci. USA 94:10833-10837; Macfarlane et al.(1997) Immunology 91:586-593; Schwartz et al. (1997) J. Clin. Invest.100:68-73; Stein et al. (1997) Antisense Technology, Ch. 11 pp. 241-264,C. Lichtenstein and W. Nellen, Eds., IRL Press; Wooldridge et al. (1997)Blood 89:2994-2998; Leclerc et al. (1997) Cell. Immunol. 179:97-106;Kline et al. (1997) J. Invest. Med. 45(3):282A; Yi et al. (1998a) J.Immunol. 160:1240-1245; Yi et al. (1998b) J. Immunol. 160:4755-4761; Yiet al. (1998c) J. Immunol. 160:5898-5906; Yi et al. (1998d) J. Immunol.161:4493-4497; Krieg (1998) Applied Antisense Oligonucleotide TechnologyCh. 24, pp. 431-448, C. A. Stein and A. M. Krieg, Eds., Wiley-Liss,Inc.; Krieg et al. (1998a) Trends Microbiol. 6:23-27; Krieg et al.(1998b) J. Immunol. 161:2428-2434; Krieg et al. (1998c) Proc. Natl.Acad. Sci. USA 95:12631-12636; Spiegelberg et al. (1998) Allergy53(455):93-97; Horner et al. (1998) Cell Immunol. 190:77-82; Jakob etal. (1998) J. Immunol. 161:3042-3049; Redford et al. (1998) J. Immunol.161:3930-3935; Weeratna et al. (1998) Antisense & Nucleic Acid DrugDevelopment 8:351-356; McCluskie et al. (1998) J. Immunol.161(9):4463-4466; Gramzinski et al. (1998) Mol. Med. 4:109-118; Liu etal. (1998) Blood 92:3730-3736; Moldoveanu et al. (1998) Vaccine 16:1216-1224; Brazolot Milan et al. (1998) Proc. Natl. Acad. Sci. USA95:15553-15558; Briode et al. (1998) J. Immunol. 161:7054-7062; Briodeet al. (1999) Int. Arch. Allergy Immunol. 118:453-456; Kovarik et al.(1999) J. Immunol. 162:1611-1617; Spiegelberg et al. (1999) Pediatr.Pulmonol. Suppl. 18:118-121; Martin-Orozco et al. (1999) Int. Immunol.11:1111-1118; EP 468,520; WO 96/02555; WO 97/28259; WO 98/16247; WO98/18810; WO 98/37919; WO 98/40100; WO 98/52581; WO 98/55495; WO98/55609 and WO 99/11275. See also Elkins et al. (1999) J. Immunol.162:2291-2298, WO 98/52962, WO 99/33488, WO 99/33868, WO 99/51259 and WO99/62923. See also Zimmermann et al. (1998) J. Immunol. 160:3627-3630;Krieg (1999) Trends Microbiol. 7:64-65 and U.S. Pat. Nos. 5,663,153,5,723,335 and 5,849,719. See also Liang et al. (1996) J. Clin. Invest.98:1119-1129; Bohle et al. (1999) Eur. J. Immunol. 29:2344-2353 and WO99/56755. See also WO 99/61056; WO 00/06588; WO 00/16804; WO 00/21556;WO 00/54803; WO 00/61151; WO 00/67023; WO 00/67787 and U.S. Pat. No.6,090,791. See also Manzel et al. (1999) Antisense Nucl. Acid Drug Dev.9:459-464; Verthelyi et al. (2001) J. Immunol. 166:2372-2377; WO01/15726; WO 01/12223; WO 01/22972; WO 01/22990; WO 01/35991; WO01/51500; WO 01/54720; U.S. Pat. Nos. 6,174,872, 6,194,388, 6,207,646,6,214,806, 6,218,371, 6,239,116. See also, WO 01/12804; WO 01/45750; WO01/55341; WO 01/55370; WO 01/62207; WO 01/68077; WO 01/68078; WO01/68103; WO 01/68116; WO 01/68117; WO 01/68143; WO 01/68144; WO01/72123; WO 01/76642; WO 01/83503; WO 01/93902; WO 02/026757; WO02/052002; WO 02/069369; WO 02/074922; U.S. Pat. Nos. 6,339,068,6,406,705, 6,426,334, 6,426,336, 6,429,199, 6,476,000.

Immunomodulatory polynucleotides generally include a CG sequence.Nucleotides flanking the CG of an IMP also appear to play a role in theimmunomodulatory activity of the polynucleotide. There remains a needfor continued identification of immunomodulatory polynucleotides.

All patents, patent applications, and publications cited herein arehereby incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

The invention relates to immunomodulatory polynucleotides (IMP) andmethods for modulating immune responses in individuals using thesepolynucleotides, particularly humans.

In one aspect, the invention provides immunomodulatory polynucleotides.In certain embodiments, the invention includes compositions whichcomprise any of the immunomodulatory polynucleotides described herein.The compositions may also include, for example, a pharmaceuticallyacceptable excipient or any of a number of other components, such as anantigen.

In one aspect, the immunomodulatory polynucleotide of the inventioncomprises (a) a palindromic sequence comprising at least two CGdinucleotides, wherein the CG dinucleotides are separated by 0, 1, 2, 3,4 or 5 bases and wherein the palindromic sequence is at least 8 bases inlength; and (b) a (TCG)_(y), wherein y is 1 or 2, wherein the 5′ T ofthe (TCG)_(y) is positioned 0, 1, 2 or 3 bases from the 5′ end of thepolynucleotide and wherein the (TCG)_(y) is separated from the 5′ end ofthe palindromic sequence by 0, 1, or 2 bases. In some immunomodulatorypolynucleotides of the invention, whether described in this paragraph orelsewhere in this application, the palindromic sequence has a basecomposition of less than two-thirds G's and C's. In some embodiments,the palindromic sequence has a base composition of greater thanone-third A's and T's.

In another aspect, the immunomodulatory polynucleotide of the inventioncomprises (a) a palindromic sequence comprising at least two CGdinucleotides, wherein the CG dinucleotides are separated by 0, 1, 2, 3,4 or 5 bases and wherein the palindromic sequence is at least 8 bases inlength; and (b) a (TCG)_(y) sequence, wherein y is 1 or 2, wherein the5′ T of the (TCG)_(y) sequence is positioned 0, 1, 2 or 3 bases from the5′ end of the polynucleotide, and further wherein the palindromicsequence of (a) includes all or part of the (TCG)_(y) sequence andwherein a CG of the (TCG)_(y) sequence may be one of the CGdinucleotides of the palindromic sequence of (a).

In another aspect, the immunomodulatory polynucleotide of the inventioncomprises (a) 5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGX₂′X₁′(CG)_(p))_(z)(SEQ ID NO: 156) wherein N are nucleosides, x=0-3, y=1-4, w=−2, −1, 0, 1or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, X₁ and X₁′ areself-complimentary nucleosides, X₂ and X₂′ are self-complimentarynucleosides, and wherein the 5′ T of the (TCG(N_(q)))_(y) sequence is0-3 bases from the 5′ end of the polynucleotide; and (b) a palindromicsequence at least 8 bases in length wherein the palindromic sequencecomprises the first (X₁X₂CGX₂′X₁′) of the (X₁X₂CGX₂′X₁′(CG)_(p)),sequences. In some embodiments, X₁ and X₂ are each either A or T.

In another aspect, the immunomodulatory polynucleotide of the inventioncomprises (a)5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGX₃X₃′CGX₂′X₁′(CG)_(p))_(z) (SEQ IDNO: 159) wherein N are nucleosides, x=0-3, y=1-4, w=−2, −1, 0, 1 or 2,p=0 or 1, q=0, 1 or 2, and z=1-20, X₁ and X₁′ are self-complimentarynucleosides, X₂ and X₂′ are self-complimentary nucleosides, X₃ and X₃′are self-complimentary nucleosides and wherein the 5′ T of the(TCG(N_(g)))₃, sequence is 0-3 bases from the 5′ end of thepolynucleotide; and (b) a palindromic sequence at least 10 bases inlength wherein the palindromic sequence comprises the first(X₁X₂CGX₃X₃′CGX₂′X₁′) (SEQ ID NO:216) of the (X₁X₂CGX₃X₃′CGX₂′X₁′(CG)_(p))_(z) (SEQ ID NO:217) sequences. In some embodiments, when p=1,X₁, X₂, and X₃ are each either A or T. In some embodiments, when p=0, atleast two of X₁, X₂, and X₃ are either A or T.

In another aspect, the immunomodulatory polynucleotide of the inventioncomprises (a)5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p))_(z)(SEQ ID NO:160) wherein N are nucleosides, x=0-3, y=1-4, w=−3, −2, −1,0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, X₁ and X₁′ areself-complimentary nucleosides, X₂ and X₂′ are self-complimentarynucleosides, X₃ and X₃′ are self-complimentary nucleosides, X₄ and X₄′are self-complimentary nucleosides, X₅ and X₅′ are self-complimentarynucleosides, and wherein the 5′ T of the (TCG(N_(g)))₃, sequence is 0-3bases from the 5′ end of the polynucleotide; and (b) a palindromicsequence at least 12 bases in length wherein the palindromic sequencecomprises the first (X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′) (SEQ ID NO:218) of the(X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p)), (SEQ ID NO:219) sequences. Insome embodiments, at least three of X₁, X₂, X₃, X₄, and X₅ are either Aor T.

In another aspect, the immunomodulatory polynucleotide of the inventioncomprises (a) 5′-N_(x)(TCG(N_(q)))_(y)N_(w)(CGX₁X₁′CG(CG)_(p))_(z) (SEQID NO:161) wherein N are nucleosides, x=0-3, y=1-4, w=−2, 0, 1 or 2, p=0or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′ are self-complimentarynucleosides and wherein the 5′ T of the (TCG(N_(q)))_(y) sequence is 0-3bases from the 5′ end of the polynucleotide; and (b) a palindromicsequence at least 8 bases in length wherein the palindromic sequencecomprises the first (CGX₁X₁′CG) of the (CGX₁X₁′CG(CG)_(p))_(z)sequences.

In another aspect, the immunomodulatory polynucleotide of the inventioncomprises (a) 5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁CGCGX₁′(CG)_(p))_(z) (SEQID NO:162) wherein N are nucleosides, x=0-3, y=1-4, w=−1, 0, 1 or 2, p=0or 1, q=0, 1 or 2, and z=1-20, X₁ and X₁′ are self-complimentarynucleosides and wherein the 5′ T of the (TCG(N_(q)))_(z) sequence is 0-3bases from the 5′ end of the polynucleotide; and (b) a palindromicsequence at least 8 bases in length wherein the palindromic sequencecomprises the first (X₁CGCGX₁′) of the (X₁CGCGX₁′(CG)_(p))_(z)sequences.

In another aspect, the immunomodulatory polynucleotide of the inventioncomprises (a)5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGCGX₃′X₂′X₁′(CG)_(p))_(z) (SEQ IDNO:163) wherein N are nucleosides, x=0-3, y=1-4, w=−2, −1, 0, 1 or 2,p=0 or 1, q=0, 1 or 2, and z=1-20, X₁ and X₁′ are self-complimentarynucleosides, X₂ and X₂′ are self-complimentary nucleosides, and whereinthe 5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ endof the polynucleotide; and (b) a palindromic sequence at least 8 basesin length wherein the palindromic sequence comprises the first(X₁X₂CGCGX₂′X₁′) of the (X₁X₂CGCGX₂′X₁′(CG)_(p))_(z) (SEQ ID NO:220)sequences. In some embodiments, X₁ and X₂ are each either A or T.

In another aspect, the immunomodulatory polynucleotide of the inventioncomprises (a)5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p))_(z) (SEQ IDNO:164) wherein N are nucleosides, x=0-3, y=1-4, w=−3, −2, −1, 0, 1 or2, p=0 or 1, q=0, 1 or 2, and z=1-20, X₁ and X₁′ are self-complimentarynucleosides, X₂ and X₂′ are self-complimentary nucleosides, X₃ and X₃′are self-complimentary nucleosides, and wherein the 5′ T of the(TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ end of thepolynucleotide; and (b) a palindromic sequence at least 10 bases inlength wherein the palindromic sequence comprises the first(X₁X₂X₃CGCGX₃′X₂′X₁′) (SEQ ID NO:221) of the(X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p))_(z) (SEQ ID NO:222) sequences. In someembodiments, when p=1, X₁, X₂, and X₃ are each either A or T. In someembodiments, when p=0, at least two of X₁, X₂, and X₃ are either A or T.

In another aspect, the immunomodulatory polynucleotide of the inventioncomprises a) 5′-N_(x)(TCG(N_(q)))_(y)N_(w)(CGX₁X₂X₂′X₁′CG(CG)_(p))_(z)(SEQ ID NO:165) wherein N are nucleosides, x=0-3, y=1-4, w=−2, 0, 1 or2, p=0 or 1, q=0, 1 or 2, and z=1-20, X₁ and X₁′ are self-complimentarynucleosides, X₂ and X₂′ are self-complimentary nucleosides, and whereinthe 5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ endof the polynucleotide; and (b) a palindromic sequence at least 8 basesin length wherein the palindromic sequence comprises the first(CGX₁X₂X₂′X₁′CG) of the (CGX₁X₂X₂′X₁′CG(CG)_(p))_(z) (SEQ ID NO:223)sequences. In some embodiments, X₁ and X₂ are each either A or T.

In another aspect, the invention provides methods of modulating animmune response in an individual, comprising administering to anindividual an immunomodulatory polynucleotide of the invention in anamount sufficient to modulate an immune response in said individual.Immunomodulation according to the methods of the invention may bepracticed on individuals including those suffering from a disorderassociated with a Th2-type immune response (e.g., allergies,allergy-induced asthma, or atopic dermatitis), individuals receivingvaccines such as therapeutic vaccines (e.g., vaccines comprising anallergy epitope, a mycobacterial epitope, or a tumor associated epitope)or prophylactic vaccines, individuals with cancer and individuals havingan infectious disease.

In another aspect, the invention provides methods of increasinginterferon-gamma (IFN-γ) in an individual, comprising administering aneffective amount of an immunomodulatory polynucleotide of the inventionto said individual. Administration of an immunomodulatory polynucleotidein accordance with the invention increases IFN-γ in the individual.

In another aspect, the invention provides methods of increasinginterferon-alpha (IFN-α) in an individual, comprising administering aneffective amount of an immunomodulatory polynucleotide of the inventionto said individual. Administration of an immunomodulatory polynucleotidein accordance with the invention increases IFN-α in the individual.

In another aspect, the invention provides methods of ameliorating one ormore symptoms of an infectious disease, comprising administering aneffective amount of an immunomodulatory polynucleotide of the inventionto an individual having an infectious disease. Administration of animmunomodulatory polynucleotide in accordance with the inventionameliorates one or more symptoms of the infectious disease.

In another aspect, the invention provides methods of ameliorating one ormore symptoms of an IgE-related disorder, comprising administering aneffective amount of an immunomodulatory polynucleotide of the inventionto an individual having an IgE-related disorder. Administration of animmunomodulatory polynucleotide in accordance with the inventionameliorates one or more symptoms of the IgE-related disorder.

The invention further relates to kits, preferably for carrying out themethods of the invention. The kits of the invention generally comprisean immunomodulatory polynucleotide of the invention (generally in asuitable container), and may further include instructions for use of theimmunomodulatory polynucleotide in immunomodulation of an individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the amount of IFN-α produced (pg/ml) fromhuman PBMCs in response to varying doses of four different IMPs: SEQ IDNOs: 1, 27, 113 and 172.

FIG. 2 contains graphs depicting NK cell lytic activity stimulated byIMPs.

MODES FOR CARRYING OUT THE INVENTION

We have discovered immunomodulatory polynucleotides and methods formodulating immune responses in individuals, particularly humans, usingthese immunomodulatory polynucleotides. The compositions of theinvention comprise an immunomodulatory polynucleotide as describedherein. The immunomodulatory polynucleotides of the invention include a)a palindromic sequence at least 8 bases in length which contains atleast one CG dinucleotide and b) at least one TCG trinucleotide at ornear the 5′ end of the polynucleotide.

We have found that immunomodulatory polynucleotides of the inventionefficiently modulate immune cells, including human cells, in a varietyof ways. We have observed that immunomodulatory polynucleotides of theinvention can effectively stimulate cytokine, including type Iinterferons, such as IFN-α and IFN-ω, and IFN-γ, production from humancells. We have also observed that immunomodulatory polynucleotides ofthe invention can effectively stimulate B cells to proliferate. We haveobserved that some of the immunomodulatory polynucleotides of theinvention activate plasmacytoid dendritic cells to undergo maturation.We have also observed that the presence of some of the immunomodulatorypolynucleotides of the invention can result in retardation ofplasmacytoid dendritic cell apoptosis in culture.

The invention also provides methods for modulating an immune response inan individual by administering an immunomodulatory polynucleotide of theinvention to the individual. Further provided are kits comprising theIMPs of the invention. The kits may further comprise instructions foradministering an immunomodulatory polynucleotide of the invention forimmunomodulation in a subject and immunomodulatory polynucleotides.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture(R. I. Freshney, ed., 1987); Handbook of Experimental Immunology (D. M.Weir & C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells(J. M. Miller & M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); The Immunoassay Handbook (D. Wild,ed., Stockton Press NY, 1994); Bioconjugate Techniques (Greg T.Hermanson, ed., Academic Press, 1996); and Methods of ImmunologicalAnalysis (R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim:VCH Verlags gesellschaft mbH, 1993).

DEFINITIONS

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. For example, “an” IMP includesone or more IMP.

As used interchangeably herein, the terms “polynucleotide” and“oligonucleotide” include single-stranded DNA (ssDNA), double-strandedDNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA(dsRNA), modified oligonucleotides and oligonucleosides or combinationsthereof. The oligonucleotide can be linearly or circularly configured,or the oligonucleotide can contain both linear and circular segments.Oligonucleotides are polymers of nucleosides joined, generally, throughphosphodiester linkages, although alternate linkages, such asphosphorothioate esters may also be used in oligonucleotides. Anucleoside consists of a purine (adenine (A) or guanine (G) orderivative thereof) or pyrimidine (thymine (T), cytosine (C) or uracil(U), or derivative thereof) base bonded to a sugar. The four nucleosideunits (or bases) in DNA are called deoxyadenosine, deoxyguanosine,deoxythymidine, and deoxycytidine. A nucleotide is a phosphate ester ofa nucleoside.

The term “immunomodulatory polynucleotide” or “IMP” as used hereinrefers to a polynucleotide that effects and/or contributes to ameasurable immune response as measured in vitro, in vivo and/or ex vivo.Examples of measurable immune responses include, but are not limited to,antigen-specific antibody production, secretion of cytokines, activationor expansion of lymphocyte populations such as NK cells, CD4+ Tlymphocytes, CD8+ T lymphocytes, B lymphocytes, and the like.Preferably, the IMP sequences preferentially activate a Th1-typeresponse.

The term “immunomodulatory” or “modulating an immune response” as usedherein includes immunostimulatory as well as immunosuppressive effects.Immunomodulation is primarily a qualitative alteration in an overallimmune response, although quantitative changes may also occur inconjunction with immunomodulation. An immune response that isimmunomodulated according to the present invention is one that isshifted towards a “Th1-type” immune response, as opposed to a “Th2-type”immune response. Th1-type responses are typically considered cellularimmune system (e.g., cytotoxic lymphocytes) responses, while Th2-typeresponses are generally “humoral”, or antibody-based. Th1-type immuneresponses are normally characterized by “delayed-type hypersensitivity”reactions to an antigen, and can be detected at the biochemical level byincreased levels of Th1-associated cytokines such as IFN-γ, IFN-α, IL-2,IL-12, and TNF-β, as well as IL-6, although IL-6 may also be associatedwith Th2-type responses as well. Th1-type immune responses are generallyassociated with the production of cytotoxic lymphocytes (CTLs) and lowlevels or transient production of antibody. Th2-type immune responsesare generally associated with higher levels of antibody production,including IgE production, an absence of or minimal CTL production, aswell as expression of Th2-associated cytokines such as IL-4.Accordingly, immunomodulation in accordance with the invention may berecognized by, for example, an increase in IFN-γ and/or IFN-α and/or adecrease in IgE production in an individual treated in accordance withthe methods of the invention as compared to the absence of treatment.

The term “3′” generally refers to a region or position in apolynucleotide or oligonucleotide 3′ (downstream) from another region orposition in the same polynucleotide or oligonucleotide. The term “3′end” refers to the 3′ terminus of the polynucleotide.

The term “5′” generally refers to a region or position in apolynucleotide or oligonucleotide 5′ (upstream) from another region orposition in the same polynucleotide or oligonucleotide. The term “5′end” refers to the 5′ terminus of the polynucleotide.

A region, portion, or sequence which is “adjacent” to another sequencedirectly abuts that region, portion, or sequence. For example, anadditional polynucleotide sequence (e.g., a TCG trinucleotide) which isadjacent to a particular portion of an immunomodulatory polynucleotidedirectly abuts that region.

The term “palindromic sequence” or “palindrome” refers to a nucleic acidsequence that is an inverted repeat, e.g., ABCDD′C′B′A′, where thebases, e.g., A, and A′, B and B′, C and C′, D and D′, are capable offorming the Watson-Crick base pairs. Such sequences may besingle-stranded or may form double-stranded structures or may formhairpin loop structures under some conditions. For example, as usedherein, “an 8 base palindrome” refers to a nucleic acid sequence inwhich the palindromic sequence is 8 bases in length, such asABCDD′C′B′A′. A palindromic sequence may be part of a polynucleotidewhich also contains non-palindromic sequences. A polynucleotide maycontain one or more palindromic sequence portions and one or morenon-palindromic sequence portions. Alternatively, a polynucleotidesequence may be entirely palindromic. In a polynucleotide with more thanone palindromic sequence portions, the palindromic sequence portions mayoverlap with each other or the palindromic sequence portions may notoverlap with each other.

The term “conjugate” refers to a complex in which an IMP and an antigenare linked Such conjugate linkages include covalent and/or non-covalentlinkages.

The term “antigen” means a substance that is recognized and boundspecifically by an antibody or by a T cell antigen receptor. Antigenscan include peptides, proteins, glycoproteins, polysaccharides, complexcarbohydrates, sugars, gangliosides, lipids and phospholipids; portionsthereof and combinations thereof. The antigens can be those found innature or can be synthetic. Antigens suitable for administration withIMP include any molecule capable of eliciting a B cell or T cellantigen-specific response. Preferably, antigens elicit an antibodyresponse specific for the antigen. Haptens are included within the scopeof “antigen.” A hapten is a low molecular weight compound that is notimmunogenic by itself but is rendered immunogenic when conjugated withan immunogenic molecule containing antigenic determinants. Smallmolecules may need to be haptenized in order to be rendered antigenic.Preferably, antigens of the present invention include peptides, lipids(e.g., sterols, fatty acids, and phospholipids), polysaccharides such asthose used in Hemophilus influenza vaccines, gangliosides andglycoproteins.

“Adjuvant” refers to a substance which, when added to an immunogenicagent such as antigen, nonspecifically enhances or potentiates an immuneresponse to the agent in the recipient host upon exposure to themixture.

The term “peptide” are polypeptides that are of sufficient length andcomposition to effect a biological response, e.g., antibody productionor cytokine activity whether or not the peptide is a hapten. Typically,the peptides are at least six amino acid residues in length. The term“peptide” further includes modified amino acids (whether or notnaturally or non-naturally occurring), such modifications including, butnot limited to, phosphorylation, glycosylation, pegylation, lipidizationand methylation.

“Antigenic peptides” can include purified native peptides, syntheticpeptides, recombinant proteins, crude protein extracts, attenuated orinactivated viruses, cells, micro-organisms, or fragments of suchpeptides. An “antigenic peptide” or “antigen polypeptide” accordinglymeans all or a portion of a polypeptide which exhibits one or moreantigenic properties. Thus, for example, an “Amb a 1 antigenicpolypeptide” or “Amb a 1 polypeptide antigen” is an amino acid sequencefrom Amb a 1, whether the entire sequence, a portion of the sequence,and/or a modification of the sequence, which exhibits an antigenicproperty (i.e., binds specifically to an antibody or a T cell receptor).

A “delivery molecule” or “delivery vehicle” is a chemical moiety whichfacilitates, permits, and/or enhances delivery of an immunomodulatorypolynucleotide to a particular site and/or with respect to particulartiming. A delivery vehicle may or may not additionally stimulate animmune response.

An “allergic response to antigen” means an immune response generallycharacterized by the generation of eosinophils and/or antigen-specificIgE and their resultant effects. As is well-known in the art, IgE bindsto IgE receptors on mast cells and basophils. Upon later exposure to theantigen recognized by the IgE, the antigen cross-links the IgE on themast cells and basophils causing degranulation of these cells,including, but not limited, to histamine release. It is understood andintended that the terms “allergic response to antigen”, “allergy”, and“allergic condition” are equally appropriate for application of some ofthe methods of the invention. Further, it is understood and intendedthat the methods of the invention include those that are equallyappropriate for prevention of an allergic response as well as treating apre-existing allergic condition.

As used herein, the term “allergen” means an antigen or antigenicportion of a molecule, usually a protein, which elicits an allergicresponse upon exposure to a subject. Typically the subject is allergicto the allergen as indicated, for instance, by the wheal and flare testor any method known in the art. A molecule is said to be an allergeneven if only a small subset of subjects exhibit an allergic (e.g., IgE)immune response upon exposure to the molecule. A number of isolatedallergens are known in the art. These include, but are not limited to,those provided in Table 1 herein.

The term “desensitization” refers to the process of the administrationof increasing doses of an allergen to which the subject has demonstratedsensitivity. Examples of allergen doses used for desensitization areknown in the art, see, for example, Formadley (1998) Otolaryngol. Clin.North Am. 31:111-127.

“Antigen-specific immunotherapy” refers to any form of immunotherapywhich involves antigen and generates an antigen-specific modulation ofthe immune response. In the allergy context, antigen-specificimmunotherapy includes, but is not limited to, desensitization therapy.

The term “microcarrier” refers to a particulate composition which isinsoluble in water and which has a size of less than about 150, 120 or100 μm, preferably less than about 50-60 μm, preferably less than about10 μm, preferably less than about 5, 2.5, 2 or 1.5 μm. Microcarriersinclude “nanocarriers”, which are microcarriers having a size of lessthan about 1 μm, preferably less than about 500 nm. Microcarriersinclude solid phase particles such as particles formed frombiocompatible naturally occurring polymers, synthetic polymers orsynthetic copolymers, although microcarriers formed from agarose orcross-linked agarose may be included or excluded from the definition ofmicrocarriers herein as well as other biodegradable materials known inthe art. Microcarriers for use in the instant invention may bebiodegradable or nonbiodegradable. Nonbiodegradable solid phasemicrocarriers are formed from polymers or other materials which arenon-erodible and/or non-degradable under mammalian physiologicalconditions, such as polystyrene, polypropylene, silica, ceramic,polyacrylamide, gold, latex, hydroxyapatite, dextran, and ferromagneticand paramagnetic materials. Biodegradable solid phase microcarriers maybe formed from polymers which are degradable (e.g., poly(lactic acid),poly(glycolic acid) and copolymers thereof) or erodible (e.g.,poly(ortho esters such as3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU) orpoly(anhydrides), such as poly(anhydrides) of sebacic acid) undermammalian physiological conditions. Microcarriers may also be liquidphase (e.g., oil or lipid based), such liposomes, iscoms(immune-stimulating complexes, which are stable complexes ofcholesterol, phospholipid and adjuvant-active saponin) without antigen,or droplets or micelles found in oil-in-water or water-in-oil emulsions.Biodegradable liquid phase microcarriers typically incorporate abiodegradable oil, a number of which are known in the art, includingsqualene and vegetable oils. Microcarriers are typically spherical inshape, but microcarriers which deviate from speherical shape are alsoacceptable (e.g., ellipsoidal, rod-shaped, etc.). Due to their insolublenature (with respect to water), microcarriers are filterable from waterand water-based (aqueous) solutions.

The term “nonbiodegradable”, as used herein, refers to a microcarrierwhich is not degraded or eroded under normal mammalian physiologicalconditions. Generally, a microcarrier is considered nonbiodegradable ifit not degraded (i.e., loses less than 5% of its mass or average polymerlength) after a 72 hour incubation at 37° C. in normal human serum.

A microcarrier is considered “biodegradable” if it is degradable orerodable under normal mammalian physiological conditions. Generally, amicrocarrier is considered biodegradable if it is degraded (i.e., losesat least 5% of its mass or average polymer length) after a 72 hourincubation at 37° C. in normal human serum.

The “size” of a microcarrier is generally the “design size” or intendedsize of the particles stated by the manufacturer. Size may be a directlymeasured dimension, such as average or maximum diameter, or may bedetermined by an indirect assay such as a filtration screening assay.Direct measurement of microcarrier size is typically carried out bymicroscopy, generally light microscopy or scanning electron microscopy(SEM), in comparison with particles of known size or by reference to amicrometer. As minor variations in size arise during the manufacturingprocess, microcarriers are considered to be of a stated size ifmeasurements show the microcarriers are ± about 5-10% of the statedmeasurement. Size characteristics may also be determined by dynamiclight scattering or obscuration techniques. Alternately, microcarriersize may be determined by filtration screening assays. A microcarrier isless than a stated size if at least 97% of the particles pass through a“screen-type” filter (i.e., a filter in which retained particles are onthe surface of the filter, such as polycarbonate or polyethersulfonefilters, as opposed to a “depth filter” in which retained particleslodge within the filter) of the stated size. A microcarrier is largerthan a stated size if at least about 97% of the microcarrier particlesare retained by a screen-type filter of the stated size. Thus, at leastabout 97% microcarriers of about 10 μm to about 10 nm in size passthrough a 10 μm pore screen filter and are retained by a 10 nm screenfilter.

As above discussion indicates, reference to a size or size range for amicrocarrier implicitly includes approximate variations andapproximations of the stated size and/or size range. This is reflectedby use of the term “about” when referring to a size and/or size range,and reference to a size or size range without reference to “about” doesnot mean that the size and/or size range is exact.

The term “immunomodulatory polynucleotide/microcarrier complex” or“IMP/MC complex” refers to a complex of an immunomodulatorypolynucleotide and a microcarrier. The components of the complex may becovalently or non-covalently linked. Non-covalent linkages may bemediated by any non-covalent bonding force, including by hydrophobicinteraction, ionic (electrostatic) bonding, hydrogen bonds and/or vander Waals attractions. In the case of hydrophobic linkages, the linkageis generally via a hydrophobic moiety (e.g., cholesterol) covalentlylinked to the IMP.

An “individual” is a vertebrate, such as avian, and is preferably amammal, more preferably a human. Mammals include, but are not limitedto, humans, primates, farm animals, sport animals, rodents and pets.

An “effective amount” or a “sufficient amount” of a substance is thatamount sufficient to effect beneficial or desired results, includingclinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. In the context of administering acomposition that modulates an immune response to a co-administeredantigen, an effective amount of an immunomodulatory polynucleotide andantigen is an amount sufficient to achieve such a modulation as comparedto the immune response obtained when the antigen is administered alone.An effective amount can be administered in one or more administrations.

The term “co-administration” as used herein refers to the administrationof at least two different substances sufficiently close in time tomodulate an immune response. Preferably, co-administration refers tosimultaneous administration of at least two different substances.

“Stimulation” of a response or parameter includes eliciting and/orenhancing that response or parameter. For example, “stimulation” of animmune response, such as Th1 response, means an increase in theresponse, which can arise from eliciting and/or enhancement of aresponse. Similarly, “stimulation” of a cytokine or cell type (such asCTLs) means an increase in the amount or level of cytokine or cell type.B cell “stimulation” includes, for example, enhanced B cellproliferation, induced B cell activation and/or increased production ofcytokines, such as IL-6 and/or TNF-α, from the stimulated B cell.

An “IgE associated disorder” is a physiological condition which ischaracterized, in part, by elevated IgE levels, which may or may not bepersistent. IgE associated disorders include, but are not limited to,allergy and allergic reactions, food allergies, allergy-relateddisorders (described below), asthma, rhinitis, atopic dermatitis,conjunctivitis, urticaria, shock, Hymenoptera sting allergies, and drugallergies, and parasite infections. The term also includes relatedmanifestations of these disorders. Generally, IgE in such disorders isantigen-specific.

An “allergy-related disorder” means a disorder resulting from theeffects of an antigen-specific IgE immune response. Such effects caninclude, but are not limited to, hypotension and shock. Anaphylaxis isan example of an allergy-related disorder during which histaminereleased into the circulation causes vasodilation as well as increasedpermeability of the capillaries with resultant marked loss of plasmafrom the circulation. Anaphylaxis can occur systemically, with theassociated effects experienced over the entire body, and it can occurlocally, with the reaction limited to a specific target tissue or organ.

The term “viral disease”, as used herein, refers to a disease which hasa virus as its etiologic agent. Examples of viral diseases includehepatitis B, hepatitis C, influenza, acquired immunodeficiency syndrome(AIDS), and herpes zoster.

As used herein, and as well-understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation or amelioration ofone or more symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, preventing spread of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

“Palliating” a disease or disorder means that the extent and/orundesirable clinical manifestations of a disorder or a disease state arelessened and/or time course of the progression is slowed or lengthened,as compared to not treating the disorder. Especially in the allergycontext, as is well understood by those skilled in the art, palliationmay occur upon modulation of the immune response against an allergen(s).Further, palliation does not necessarily occur by administration of onedose, but often occurs upon administration of a series of doses. Thus,an amount sufficient to palliate a response or disorder may beadministered in one or more administrations.

An “antibody titer”, or “amount of antibody”, which is “elicited” by animmunomodulatory polynucleotide and antigen refers to the amount of agiven antibody measured at a time point after administration ofimmunomodulatory polynucleotide and antigen.

A “Th1-associated antibody” is an antibody whose production and/orincrease is associated with a Th1 immune response. For example, IgG2a isa Th1-associated antibody in mouse. For purposes of this invention,measurement of a Th1-associated antibody can be measurement of one ormore such antibodies. For example, in human, measurement of aTh1-associated antibody could entail measurement of IgG1 and/or IgG3.

A “Th2-associated antibody” is an antibody whose production and/orincrease is associated with a Th2 immune response. For example, IgG1 isa Th2-associated antibody in mouse. For purposes of this invention,measurement of a Th2-associated antibody can be measurement of one ormore such antibodies. For example, in human, measurement of aTh2-associated antibody could entail measurement of IgG2 and/or IgG4.

To “suppress” or “inhibit” a function or activity, such as cytokineproduction, antibody production, or histamine release, is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, a composition comprising animmunomodulatory polynucleotide and antigen which suppresses histaminerelease reduces histamine release as compared to, for example, histaminerelease induced by antigen alone. As another example, a compositioncomprising an immunomodulatory polynucleotide and antigen whichsuppresses antibody production reduces extent and/or levels of antibodyas compared to, for example, extent and/or levels of antibody producedby antigen alone.

A “serum protein” is a protein that is normally found in the serum ofdisease-free mammals, particularly disease-free bovines. The mostprevalent serum protein is serum albumin.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

Compositions of the Invention

The invention provides immunomodulatory polynucleotides (IMPs) formodulating immune responses in individuals. Compositions of theinvention comprise an immunomodulatory polynucleotide alone (or acombination of two or more immunomodulatory polynucleotides) or inconjunction with another immunomodulatory agent, such as a peptide, anantigen (described below) and/or an additional adjuvant. Compositions ofthe invention may comprise an immunomodulatory polynucleotide andpharmaceutically acceptable excipient. Pharmaceutically acceptableexcipients, including buffers, are well known in the art. Remington: TheScience and Practice of Pharmacy, 20th edition, Mack Publishing (2000).

Upon administration, compositions comprising an antigen, animmunomodulatory polynucleotide of the invention, and optionally anadjuvant can lead to a potentiation of a immune response to the antigenand thus, can result in an enhanced immune response compared to thatwhich results from a composition comprising the IMP and antigen alone.Adjuvants are known in the art and include, but are not limited to,oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts),liposomes and microparticles, including but not limited to, polystyrene,starch, polyphosphazene and polylactide/polyglycosides. Other suitableadjuvants also include, but are not limited to, MF59, DETOX™ (Ribi),squalene mixtures (SAF-1), muramyl peptide, saponin derivatives,mycobacterium cell wall preparations, monophosphoryl lipid A, mycolicacid derivatives, nonionic block copolymer surfactants, Quil A, choleratoxin B subunit, polyphosphazene and derivatives, and immunostimulatingcomplexes (ISCOMs) such as those described by Takahashi et al. (1990)Nature 344:873-875, as well as, lipid-based adjuvants and othersdescribed herein. For veterinary use and for production of antibodies inanimals, mitogenic components of Freund's adjuvant (both complete andincomplete) can be used.

IMPs of the invention may be combined with other therapies forparticular indications. For example, in addition to an IMP, compositionsof the invention may also comprise anti-malarial drugs such aschloroquine for malaria patients, leishmanicidal drugs such aspentamidine and/or allopurinol for leishmaniasis patients,anti-mycobacterial drugs such as isoniazid, rifampin and/or ethambutolfor tuberculosis patients, or allergen desensitization reagents foratopic (allergy) patients.

As described herein, compositions of the invention may include IMPs andmay further comprise one or more additional immunotherapeutic agents(i.e., an agent which acts via the immune system and/or is derived fromthe immune system) including, but not limited to, cytokine, adjuvantsand antibodies. Examples of therapeutic antibodies include those used inthe cancer context (e.g., anti-tumor antibodies), such as thosedescribed below.

Immunomodulatory Polynucleotides

In accordance with the present invention, the immunomodulatorypolynucleotide contains at least one palindromic sequence (i.e.,palindrome) of at least 8 bases in length containing at least one CGdinucleotide. The IMP also contains at least one TCG trinucleotidesequence at or near the 5′ end of the polynucleotide (i.e., 5′-TCG). Insome instances, the palindromic sequence and the 5′-TCG are separated by0, 1 or 2 bases in the IMP. In some instances the palindromic sequenceincludes all or part of the 5′-TCG.

IMPs have been described in the art and their activity may be readilyidentified using standard assays which indicate various aspects of theimmune response, such as cytokine secretion, antibody production, NKcell activation, B cell proliferation, T cell proliferation. See, e.g.,WO 97/28259; WO 98/16247; WO 99/11275; Krieg et al. (1995) Nature374:546-549; Yamamoto et al. (1992a); Ballas et al. (1996); Klinman etal. (1997); Sato et al. (1996); Pisetsky (1996a); Shimada et al. (1986)Jpn. J. Cancer Res. 77:808-816; Cowdery et al. (1996) J. Immunol.156:4570-4575; Roman et al. (1997); Lipford et al. (1997a); WO 98/55495and WO 00/61151. Accordingly, these and other methods can be used toidentify, test and/or confirm immunomodulatory IMPs.

The IMP can be of any length greater than 10 bases or base pairs,preferably greater than 15 bases or base pairs, more preferably greaterthan 20 bases or base pairs in length.

As is clearly conveyed herein, it is understood that, with respect toformulae described herein, any and all parameters are independentlyselected. For example, if x=0-2, y may be independently selectedregardless of the values of x (or any other selectable parameter in aformula).

In some embodiments, an IMP comprises a) a palindromic sequence at least8 bases in length which contains at least two CG dinucleotides, wherethe CG dinucleotides are separated from each other by 0, 1, 2, 3, 4 or 5bases, and b) a (TCG)_(y) sequence positioned 0, 1, 2, or 3 bases fromthe 5′ end of the polynucleotide, where y is 1 or 2, and where the 3′end of the (TCG)_(y) sequence is separated from the 5′ end of thepalindromic sequence by 0, 1 or 2 bases. In some embodiments, a CGdinucleotide of the (TCG)_(y) sequence of (b) may count for one of theat least two CG dinucleotides in the palindromic sequence of (a). Insome embodiments, the CG dinucleotides of the palindromic sequence areseparated from each other by 1, 3 or 4 bases. In some IMPs of theinvention, whether described in this paragraph or elsewhere in theapplication, the palindromic sequence has a base composition of lessthan two-thirds G's and C's. In some embodiments, the palindromicsequence has a base composition of greater than one-third A's and T's.

In some embodiments, an IMP comprises a) a palindromic sequence at least8 bases in length which contains at least two CG dinucleotides, wherethe CG dinucleotides are separated from each other by 0, 1, 2, 3, 4 or 5bases, and b) a (TCG)_(y) sequence positioned 0, 1, 2, or 3 bases fromthe 5′ end of the polynucleotide, where y is 1 or 2, where thepalindromic sequence includes all or part of the (TCG)_(y) sequence, andwhere a CG dinucleotide of the (TCG)_(y) sequence of (b) may count forone of the CG dinucleotides of the palindromic sequence of (a).Preferably, in some embodiments, the CG dinucleotides of the palindromicsequence are separated from each other by 1, 3 or 4 bases.

Accordingly, in some embodiments, an IMP may comprise a sequence of theformula: 5′-N_(x)(TCG(N_(g)))_(y)N_(w)(X₁CGX₁′(CG)_(p))_(z) (SEQ IDNO:155) wherein N are nucleosides with x=0-3, y=1-4, w=−1, 0, 1 or 2,p=0 or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′ areself-complimentary and wherein the 5′ T of the (TCG(N_(q)))_(y) sequenceis 0-3 bases from the 5′ end of the polynucleotide. The IMP furthercomprises a palindromic sequence 8 bases in length or greater whereinthe palindromic sequence comprises at least one of the (X₁CGX₁′(CG)_(p))sequences. In an IMP with w=−1, the 3′ base of the (TCG(N_(q)))_(y)sequence is the 5′ X₁ of the first (X₁CGX₁′(CG)_(p)) sequence. In someembodiments, the (TCG(N_(q)))_(y) sequence is separated from thepalindromic sequence by 0, 1 or 2 bases. In other embodiments, thepalindromic sequence includes all or part of the (TCG(N_(q)))_(y)sequence. In some embodiments, when p=0, X₁ is either A or T.

In some embodiments, the IMP comprises the following sequences(palindromic sequences underlined):

5′-TCGTCGACGTCGAGATGATAT; (SEQ ID NO: 35) 5′-TCGTCGACGTCGACGAGATAT; (SEQID NO: 60) 5′-TCGACGTCGACGTCGACGTAT; (SEQ ID NO: 61)5′-TCGGTCGACGTCGACCGATT; (SEQ ID NO: 82) 5′-TCGGACGTCGACGTCCGATT; (SEQID NO: 83) 5′-TCGACGTCGA; (SEQ ID NO: 105) 5′-TCGGACGTCGACGTGCGATT; (SEQID NO: 114) 5′-TCGACGTCGACGTCGACGTCGA; (SEQ ID NO: 119)5′-ACGTCGACGTCGACGTCGACGT; (SEQ ID NO: 120) 5′-TCGTCGACGTCGACGTCGACGT;(SEQ ID NO: 121) 5′-TCGTCGGCGCCGGCGCCGGCGC; (SEQ ID NO: 122)5′-TCGTCGCCGGCGCCGGCGCCGG; (SEQ ID NO: 123) 5′-TCGATACGTCGACGTCGACGT.(SEQ ID NO: 124)

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂X₃CGX₃′X₂′X₁′(CG)_(p))_(z) (SEQ ID NO:157) wherein N are nucleosides with x=0-3, y=1-4, w=−3, −2, −1, 0, 1 or2, p=0 or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′,and X₃ and X₃′ are self-complimentary and wherein the 5′ T of the(TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ end of thepolynucleotide. The IMP further comprises a palindromic sequence 8 basesin length or greater wherein the palindromic sequence comprises thefirst (X₁X₂X₃CGX₃′X₂′X₁′) of the at least one(X₁X₂X₃CGX₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:224) sequence. In an IMP withw=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of thefirst (X₁X₂X₃CGX₃′X₂′ X₁′(CG)_(p)) (SEQ ID NO:224) sequence. In an IMPwith w=−2, the penultimate (i.e., second to last) and the ultimate(i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁ andX₂, respectively, of the first (X₁X₂X₃CGX₃′X₂′X₁′(CG)_(p)) (SEQ IDNO:224) sequence. In an IMP with w=−3, the antepenultimate (i.e., thirdto last), the penultimate (i.e., second to last) and the ultimate (i.e.,last) 3′ bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁, X₂, andX₃, respectively, of the first (X₁X₂X₃CGX₃′X₂′X₁′(CG)_(p)) (SEQ IDNO:224) sequence. In some embodiments, the (TCG(N_(q)))_(y) sequence isseparated from the palindromic sequence by 0, 1 or 2 bases. In otherembodiments, the palindromic sequence includes all or part of the(TCG(N_(q)))_(y) sequence. In some embodiments, when p=1, X₁, X₂, and X₃are each either A or T. In some embodiments, when p=0, at least two ofX₁, X₂, and X₃ are either A or T.

In some embodiments, the IMP comprises the following sequences(palindromic sequences underlined):

5′-TCGTCGAAACGTTTCGACAGT; (SEQ ID NO: 62) 5′-TCGTCGAGACGTCTCGAC AGT;(SEQ ID NO: 63) 5′-TCGTCGAAGCGCTTCGACAGT; (SEQ ID NO: 125)5′-TCGTCGAATCGATTCGACAGT; (SEQ ID NO: 126) 5′-TCGTCGAGTCGACTCGACAGT;(SEQ ID NO: 127) 5′-TCGTCGCAACGTTGCGACAGT; (SEQ ID NO: 128)5′-TCGTCGCCGCGCGGCGACAGT; (SEQ ID NO: 129) 5′-TCGAAACGTTTCGACAGTGAT.(SEQ ID NO: 130)

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p))_(z) (SEQID NO: 158) wherein N are nucleosides with x=0-3, y=1-4, w=−3, −2, −1,0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂ andX₂′, X₃ and X₃′, and X₄ and X₄′ are self-complimentary and wherein the5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ end ofthe polynucleotide. The IMP further comprises a palindromic sequence 10bases in length or greater wherein the palindromic sequence comprisesthe first (X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′) (SEQ ID NO:225) of the at least one(X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:226) sequence. In an IMPwith w=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ ofthe first (X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:226) sequence. Inan IMP with w=−2, the penultimate (i.e., second to last) and theultimate (i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequence are the5′ X₁ and X₂, respectively, of the first(X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:226) sequence. In an IMPwith w=−3, the antepenultimate (i.e., third to last), the penultimate(i.e., second to last) and the ultimate (i.e., last) 3′ bases of the(TCG(N_(q)))_(y) sequence are the 5′ X₁, X₂, and X₃, respectively, ofthe first (X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:226) sequence. Insome embodiments, the (TCG(N_(q)))_(y) sequence is separated from thepalindromic sequence by 0, 1 or 2 bases. In other embodiments, thepalindromic sequence includes all or part of the (TCG(N_(q)))_(y)sequence. In some embodiments, when p=1, at least three of X₁, X₂, X₃,and X₄ are either A or T. In some embodiments, when p=0, at least two ofX₁, X₂, X₃, and X₄ are either A or T.

In some embodiments, the IMP comprises the following sequences(palindromic sequences underlined):

5′-TCGTCGAAAACGTTTTCGAGAT; (SEQ ID NO: 64) 5′-TCGAAAACGTTTTCGAGATGAT;(SEQ ID NO: 65) 5′-TCGAGGACGTCCTCGAGATGAT; (SEQ ID NO: 66)5′-TCGAGGTCGACCTCGAGATGAT; (SEQ ID NO: 131) 5′-ATCGATGTCGACATCGATATGAT;(SEQ ID NO: 132) 5′-TCGTCGTCGACGACGAGATGAT. (SEQ ID NO: 133)

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁CGCGX₁′(CG)_(p))_(z) (SEQ ID NO: 162)wherein N are nucleosides with x=0-3, y=1-4, w=−1, 0, 1 or 2, p=0 or 1,q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′ are self-complimentary andwherein the 5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases from the5′ end of the polynucleotide. The IMP further comprises a palindromicsequence 8 bases in length or greater wherein the palindromic sequencecomprises the first (X₁CGCGX₁′) of the at least one (X₁CGCGX₁′(CG)_(p))sequence. In an IMP with w=−1, the 3′ base of the (TCG(N_(q)))_(y)sequence is the 5′ X₁ of the first (X₁CGCGX₁′(CG)_(p)) sequence. In someembodiments, the (TCG(N_(q)))_(y) sequence is separated from thepalindromic sequence by 0, 1 or 2 bases. In other embodiments, thepalindromic sequence includes all or part of the (TCG(N_(q)))_(y)sequence. In some embodiments, the IMP comprises the following sequences(palindromic sequences underlined):

5′-TCGTCGTCGCGACGAGATGAT; (SEQ ID NO: 50) 5′-TCGTCGACGCGTCGAGATGAT; (SEQID NO: 142) 5′-TCGTCGGCGCGCCGAGATGAT. (SEQ ID NO: 143)

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(CGX₁X₁′CG(CG)_(p))_(z) (SEQ ID NO: 161)wherein N are nucleosides with x=0-3, y=1-4, w=−2, 0, 1 or 2, p=0 or 1,q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′ are self-complimentary andwherein the 5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases from the5′ end of the polynucleotide. The IMP further comprises a palindromicsequence 8 bases in length or greater wherein the palindromic sequencecomprises the first (CGX₁X₁′CG) of the at least one (CGX₁X₁′CG(CG)_(p))sequence. In an IMP with w=−2, the penultimate (i.e., second to last)and the ultimate (i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequenceare CG and are the 5′ CG of the first (CGX₁X₁′CG(CG)_(p)) sequence. Insome embodiments, the (TCG(N_(q)))_(y) sequence is separated from thepalindromic sequence by 0, 1 or 2 bases. In other embodiments, thepalindromic sequence includes all or part of the (TCG(N_(q)))_(y)sequence. In some embodiments, the IMP comprises the following sequences(palindromic sequences underlined):

5′-TCGTCGCGATCGCGAGATGAT; (SEQ ID NO: 49) 5′-TCGTCGCGTACGCGAGATGAT; (SEQID NO: 139) 5′-TCGTCGCGGCCGCGAGATGAT; (SEQ ID NO: 140)5′-TCGCGATCGCGCGATCGCGA. (SEQ ID NO: 141)

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGX₃X₃′CGX₂′X₁′(CG)_(p))_(z) (SEQ IDNO: 159) wherein N are nucleosides with x=0-3, y=1-4, w=−2, −1, 0, 1 or2, p=0 or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′,and X₃ and X₃′ are self-complimentary and wherein the 5′ T of the(TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ end of thepolynucleotide. The IMP further comprises a palindromic sequence 10bases in length or greater wherein the palindromic sequence comprisesthe first (X₁X₂CGX₃X₃′CGX₂′X₁′) (SEQ ID NO:216) of the at least one(X₁X₂CGX₃X₃′CGX₂′X₁′(CG)_(p)) (SEQ ID NO:217) sequence. In an IMP withw=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of thefirst (X₁X₂CGX₃X₃′CGX₂′X₁′(CG)_(p)) (SEQ ID NO:217) sequence. In an IMPwith w=−2, the penultimate (i.e., second to last) and the ultimate(i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁ andX₂, respectively, of the first (X₁X₂CGX₃X₃′CGX₂′X₁′ (CG)_(p)) (SEQ IDNO:217) sequence. In some embodiments, the (TCG(N_(q)))_(y) sequence isseparated from the palindromic sequence by 0, 1 or 2 bases. In otherembodiments, the palindromic sequence includes all or part of the(TCG(N_(q)))_(y) sequence. In some embodiments, when p=1, X₁, X₂, and X₃are each either A or T. In some embodiments, when p=0, at least two ofX₁, X₂, and X₃ are either A or T. In some embodiments, the IMP comprisesthe following sequences (palindromic sequences underlined):

5′-TCGGACGATCGTCGACGATCGTC; (SEQ ID NO: 86) 5′-TCGTCGGACGATCGTCACGACG;(SEQ ID NO: 87) 5′-TCGGTCGATCGACGTCGATCGAC; (SEQ ID NO: 134)5′-TCGGACGGCCGTCGACGGCCGTC; (SEQ ID NO: 135) 5′-TCGGACGTACGTCGACGTACGTC;(SEQ ID NO: 136) 5′-TCGATCGTACGATATCGTACGAT; (SEQ ID NO: 137)5′-TCGTCGGACGATCGTCCGACGA. (SEQ ID NO: 138)

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGX₂′X₁′(CG)_(p))_(z) (SEQ ID NO: 156)wherein N are nucleosides with x=0-3, y=1-4, w=−2, −1, 0, 1 or 2, p=0 or1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′ areself-complimentary, and wherein the 5′ T of the (TCG(N_(q)))_(y)sequence is 0-3 bases from the 5′ end of the polynucleotide. The IMPfurther comprises a palindromic sequence 8 bases in length or greaterwherein the palindromic sequence comprises the first (X₁X₂CGX₂′X₁′) ofthe at least one (X₁X₂CGX₂′X₁′(CG)_(p))_(z) sequence. In an IMP withw=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of thefirst (X₁X₂CGX₂′X₁′(CG)_(p)) sequence. In an IMP with w=−2, thepenultimate (i.e., second to last) and the ultimate (i.e., last) 3′bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁ and X₂,respectively, of the first (X₁X₂CGX₂′X₁′(CG)_(p)) sequence. In someembodiments, the (TCG(N_(q)))_(y) sequence is separated from thepalindromic sequence by 0, 1 or 2 bases. In other embodiments, thepalindromic sequence includes all or part of the (TCG(N_(q)))_(y)sequence. In some embodiments, X₁ and X₂ are each either A or T.

In some embodiments, the IMP comprises the following sequences(palindromic sequences underlined):

5′-TCGAACGTTCGTTCGAACGAACGTT; (SEQ ID NO: 147) 5′-TCGAACGTTTTCGAAAACGTT;(SEQ ID NO: 148) 5′-TCGTCGAACGTTCCTTAACGTTCG; (SEQ ID NO: 7)5′-TCGAACGTTAACGTTCGATT; (SEQ ID NO: 80) 5′-TCGTCGAACGTTCGAGATGAT; (SEQID NO: 27) 5′-GGTCGAACGTTCGAGGGGGG; (SEQ ID NO: 30)5′-TCGTCGAACGTTCGAGGGGGG; (SEQ ID NO: 32) 5′-TTCGAACGTTCGAACGTTCGAAT;(SEQ ID NO: 38) 5′-TCGAACGTTCGAACGTTCGAAT; (SEQ ID NO: 39)5′-TCGTCGAACGTTCGACGA; (SEQ ID NO: 52) 5′-TTTCGAACGTTCGAACGTTCGAAAT;(SEQ ID NO: 57) 5′-TTTTCGAACGTTCGAACGTTCGAAAAT; (SEQ ID NO: 58)5′-TTTTCGAACGTTCGAACGTTCGAAT; (SEQ ID NO: 59) 5′-TCGAACGTTCGAACGTTCGA;(SEQ ID NO: 97) 5′-TTCGAACGTTCGAA; (SEQ ID NO: 98)5′-TCGTCGAACGTTCGAGAT; (SEQ ID NO: 99) 5′-TCGTCGAACGTTCGAG; (SEQ ID NO:100) 5′-TCGTCGAACGTTCGA; (SEQ ID NO: 101) 5′-TCGAACGTTCGAG; (SEQ ID NO:102) 5′-TCGAACGTTCGA; (SEQ ID NO: 103) 5′-TCGAACGTTCG; (SEQ ID NO: 104)5′-TCGTCGTCGAACGTTCGAGAT; (SEQ ID NO: 106) 5′-TCGTCGTCGTCGAACGTTCGA;(SEQ ID NO: 107) 5′-TCGTCGTCGAACGTTCGACGAGAT; (SEQ ID NO: 108)5′-TCGAACGTTCGAACGTTCGAACGTT; (SEQ ID NO: 113) 5′-CTTCGAACGTTCGAAGTG;(SEQ ID NO: 115) 5′-TGATCGTCGAACGTTCGACGATCA; (SEQ ID NO: 116)5′-TCGAACGTTCGAACGTTCGAATTTT; (SEQ ID NO: 117) 5′-TCGCGAACGTTCGAACGTTCG;(SEQ ID NO: 150) 5′-TCGCGAACGTTCGAACGTTTC; (SEQ ID NO: 151)5′-TCGATAACGTTCGAACGTTAT; (SEQ ID NO: 152) 5′-TCGATAACGTTCGAACGTTTC;(SEQ ID NO: 153) 5′-TCGTCGAACGTTCGAGATG; (SEQ ID NO: 166)5′-TCGTCGAACGTTCG; (SEQ ID NO: 167) 5′-TCGAACGTTCGA TCGAACGTTCGA; (SEQID NO: 168) 5′-TCGACCGGTCGACCGGTCGA; (SEQ ID NO: 169)5′-TCGAACGTTCGAACGTTGATGT; (SEQ ID NO: 170) 5′-TCGAACGTTCGAAGATGATGAT;(SEQ ID NO: 171) 5′-TCGAACGTTCGAACGTTCGAACG; (SEQ ID NO: 175)5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT; (SEQ ID NO: 172)5′-TCGATAACGTTCGAACGTTCGAACGTTAT; (SEQ ID NO: 173)5′-TCGTAACGTTCGAACGTTCGAACGTTA. (SEQ ID NO: 174)

In some embodiments, in an IMP comprising formula of SEQ ID NO:156, X₁X₂is not AA. In some embodiments, in an IMP comprising formula of SEQ IDNO:156, X₁ is not A. Accordingly, in some embodiments, the IMP comprisesthe following sequences (palindromic sequences underlined):

(SEQ ID NO: 81) 5′-TCGAGCGCTAGCGCTCGATT; (SEQ ID NO: 82)5′-TCGGTCGACGTCGACCGATT; (SEQ ID NO: 83) 5′-TCGGACGTCGACGTCCGATT; (SEQID NO: 84) 5′-TCGTTCGAATTCGAACGATT. (SEQ ID NO: 112)5′-TCGTCGGCCGGCCGAGATGAT; (SEQ ID NO: 79) 5′-TCGGACGTCCGGACGTCCGA; (SEQID NO: 48) 5′-TCGTCGCACGTGCGAGATGAT; (SEQ ID NO: 51)5′-TCGTCGTACGTACGAGATGAT; (SEQ ID NO: 70) 5′-TCGTCGGGCGCCCGAGATGAT; (SEQID NO: 71) 5′-TCGTCGCGCGCGCGAGATGAT; (SEQ ID NO: 72)5′-TCGTCGCTCGAGCGAGATGAT; (SEQ ID NO: 73) 5′-TCGTCGCCCGGGCGAGATGAT; (SEQID NO: 74) 5′-TCGTCGTGCGCACGAGATGAT; (SEQ ID NO: 76)5′-TCGTCGTCCGGACGAGATGAT; (SEQ ID NO: 77) 5′-TCGAGCGCTCGAGCGCTCGA; (SEQID NO: 46) 5′-TCGTCGGTCGACCGAGATGAT; (SEQ ID NO: 47)5′-TCGTCGGACGTCCGAGATGAT; (SEQ ID NO: 44) 5′-TCGTCGAGCGCTCGAGATGAT; (SEQID NO: 40) 5′-TCGATTCGAACGTTCGAACGTTCG; (SEQ ID NO: 41)5′-TCGTTCGAACGTTCGAAGTGAT; (SEQ ID NO: 42) 5′-TCGTTCGAACGTTCGAACGA; (SEQID NO: 53) 5′-TCGTTCGAACGTTCGAACGTTCG; (SEQ ID NO: 54)5′-TCGTTCGAACGTTCGAA; (SEQ ID NO: 55) 5′-TCGTTCGAACGTTCGAACGTTCGAA; (SEQID NO: 56) 5′-TCGTTCGAACGTTCGAACGATTTTTCGTTCGAACGTTCGAACGA; (SEQ ID NO:43) 5′-TCGATCGATCGATCGATCGATT; (SEQ ID NO: 45) 5′-TCGTCGATCGATCGAGATGAT;(SEQ ID NO: 69) 5′-TCGTCGACCGGTCGAGATGAT; (SEQ ID NO: 75)5′-TCGTCGTTCGAACGAGATGAT; (SEQ ID NO: 78) 5′-TCGGTCGACCGGTCGACCGA; (SEQID NO: 109) 5′-TCGTTCGAACGTTCGAACGTTCGAACG; (SEQ ID NO: 118)5′-TCGTTCGAACGTTCGAACGAATGAT; (SEQ ID NO: 176)5′-TCGACCGGTCGACCGGTCGACCGGT; (SEQ ID NO: 177) 5′-TCGCGCGCGCGCGCGCGCGA;(SEQ ID NO: 178) 5′-TCGCCCGGGCGCCCGGGCGA; (SEQ ID NO: 179)5′-TCGGCCGGACGTCCGGACGA; (SEQ ID NO: 180) 5′-TCGGCCGGCCGGCCGGCCGA.

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(g)))_(y)N_(w)(X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p))_(z)(SEQ ID NO:160) wherein N are nucleosides with x=0-3, y=1-4, w=−3, −2,−1, 0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂and X₂′, X₃ and X₃′, X₄ and X₄′, and X₅ and X₅′ are self-complimentary,and wherein the 5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases fromthe 5′ end of the polynucleotide. The IMP further comprises apalindromic sequence 12 bases in length or greater wherein thepalindromic sequence comprises the first (X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′)(SEQ ID NO:218) of the at least one((X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:219) sequence. In anIMP with w=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁of the first (X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:219)sequence. In an IMP with w=−2, the penultimate (i.e., second to last)and the ultimate (i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequenceare the 5′ X₁ and X₂, respectively, of the first(X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:219) sequence. In anIMP with w=−3, the antepenultimate (i.e., third to last), thepenultimate (i.e., second to last) and the ultimate (i.e., last) 3′bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁, X₂, and X₃,respectively, of the first (X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p)) (SEQ IDNO:219) sequence. In some embodiments, the (TCG(N_(q)))_(y) sequence isseparated from the palindromic sequence by 0, 1 or 2 bases. In otherembodiments, the palindromic sequence includes all or part of the(TCG(N_(q)))_(y) sequence. In some embodiments, at least three of X₁,X₂, X₃, X₄, and X₅ are either A or T. In some embodiments, the IMPcomprises the following sequences (palindromic sequences underlined):

5′-TCGTGCATCGATGCAACG; (SEQ ID NO: 93) 5′-TCGTGCATCGATGCAGATGAT;(SEQ ID NO: 110) 5′-TCGTGCATCGATGCATGCATCGATGCA; (SEQ ID NO: 111)5′-TCGTGCATCGATGCACGA. (SEQ ID NO: 149)

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGCGX₂X₁(CG)_(p))_(z) (SEQ ID NO:163)wherein N are nucleosides with x=0-3, y=1-4, w=−2, −1, 0, 1 or 2, p=0 or1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, and X₂ and X₂′ areself-complimentary, and wherein the 5′ T of the (TCG(N_(q)))_(y)sequence is 0-3 bases from the 5′ end of the polynucleotide. The IMPfurther comprises a palindromic sequence 8 bases in length or greaterwherein the palindromic sequence comprises the first (X₁X₂CGCGX₂′X₁′) ofthe at least one (X₁X₂CGCGX₂′X₁′(CG)_(p)) (SEQ ID NO:220) sequence. Inan IMP with w=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′X₁ of the first (X₁X₂CGCGX₂′X₁′(CG)_(p)) (SEQ ID NO:220) sequence. In anIMP with w=−2, the penultimate (i.e., second to last) and the ultimate(i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁ andX₂, respectively, of the first (X₁X₂CGCGX₂′X₁′(CG)_(p)) (SEQ ID NO:220)sequence. In some embodiments, the (TCG(N_(q)))_(y) sequence isseparated from the palindromic sequence by 0, 1 or 2 bases. In otherembodiments, the palindromic sequence includes all or part of the(TCG(N_(q)))_(y) sequence. In some embodiments, X₁ and X₂ are eacheither A or T. In some embodiments, the IMP comprises the followingsequences (palindromic sequence underlined):

5′-TCGTCGATCGCGATCGACGA. (SEQ ID NO: 144)

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p))_(z) (SEQ IDNO:164) wherein N are nucleosides with x=0-3, y=1-4, w=−3, −2, −1, 0, 1or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′and X₃ and X₃′ are self-complimentary, and wherein the 5′ T of the(TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ end of thepolynucleotide. The IMP further comprises a palindromic sequence 10bases in length or greater wherein the palindromic sequence comprisesthe first (X₁X₂X₃CGCGX₃′X₂′X₁′) (SEQ ID NO:221) of the at least one(X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:222) sequence. In an IMP withw=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of thefirst (X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p)) (SEQ ID NO:222) sequence. In an IMPwith w=−2, the penultimate (i.e., second to last) and the ultimate(i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁ andX₂, respectively, of the first (X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p)) (SEQ IDNO:222) sequence. In an IMP with w=−3, the antepenultimate (i.e., thirdto last), the penultimate (i.e., second to last) and the ultimate (i.e.,last) 3′ bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁, X₂, andX₃, respectively, of the first (X₁X₂X₃CGCGX₃′X₂:X₁′ (CG)_(p)) (SEQ IDNO:222) sequence. In some embodiments, the (TCG(N_(q)))_(y) sequence isseparated from the palindromic sequence by 0, 1 or 2 bases. In otherembodiments, the palindromic sequence includes all or part of the(TCG(N_(q)))_(y) sequence. In some embodiments, when p=1, X₁, X₂, and X₃are each either A or T. In some embodiments, when p=0, at least two ofX₁, X₂, and X₃ are either A or T. In some embodiments, the IMP comprisesthe following sequences (palindromic sequence underlined):

5′-TCGTCGAATCGCGATTCGACGA. (SEQ ID NO: 145)

In some embodiments, an IMP may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(CGX₁X₂X₂′X₁′CG(CG)_(p))_(z) (SEQ ID NO:165) wherein N are nucleosides with x=0-3, y=1-4, w=−2, 0, 1 or 2, p=0or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, and X₂ and X₂′ areself-complimentary, and wherein the 5′ T of the (TCG(N_(q)))_(y)sequence is 0-3 bases from the 5′ end of the polynucleotide. The IMPfurther comprises a palindromic sequence 8 bases in length or greaterwherein the palindromic sequence comprises the first (CGX₁X₂X₂′X₁′CG) ofthe at least one (CGX₁X₂X₂′X₁′CG(CG)_(p)) (SEQ ID NO:223) sequence. Inan IMP with w=−2, the penultimate (i.e., second to last) and theultimate (i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequence are CGand are the 5′ CG of the first (CGX₁X₂X₂′X₁′CG(CG)_(p)) (SEQ ID NO:223)sequence. In some embodiments, the (TCG(N_(q)))_(y) sequence isseparated from the palindromic sequence by 0, 1 or 2 bases. In otherembodiments, the palindromic sequence includes all or part of the(TCG(N_(q)))_(y) sequence. In some embodiments, X₁ and X₂ are eacheither A or T. In some embodiments, the IMP comprises the followingsequences (palindromic sequence underlined):

5′-TCGTCGCGATATCGCGACGA. (SEQ ID NO: 146)

For IMPs comprising any of the motifs described herein (i.e., SEQ IDNOs:155-165) where y=2 or more, the (N_(q)) in each of the y repetitionsof the (TCG(N_(q))) is independently selected. For example, in an IMPwith y=2, the first TCG(N_(q)) may have N=A and q=1 and the secondTCG(N_(q)) may have q=0 in which case this portion of the IMP would be .. . TCGATCG . . . . In some embodiments of IMPs comprising any of themotifs described herein (i.e., SEQ ID NOs:155-165) in some embodiments,x is preferably 0 or 1. In some embodiments of IMPs comprising any ofthe motifs described herein (i.e., SEQ ID NOs:155-165), y is preferably1 or 2. In some embodiments of IMPs comprising any of the motifsdescribed herein (i.e., SEQ ID NOs:155-165), w is preferably 0. In someembodiments of IMPs comprising any of the motifs described herein (i.e.,SEQ ID NOs:155-165), z is preferably 1, 2, 3, 4, 5, 6, 7 or 8.

As noted above, the IMPs contain at least one the palindromic sequenceat least 8 bases in length. In some embodiments, an IMP contains atleast one palindromic sequence of at least the following lengths (inbases): 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30. In some embodiments,the palindromic sequence is repeated at least once in an IMP. In someembodiments, the palindromic sequence also includes bases 5′ of the(TCG(N_(q)))_(y) sequence, if any.

An immunomodulatory polynucleotide may contain modifications.Modifications of IMP include any known in the art, but are not limitedto, modifications of the 3′OH or 5′OH group, modifications of thenucleotide base, modifications of the sugar component, and modificationsof the phosphate group. Various such modifications are described below.Modified bases may be included in the palindromic sequence of an IMP aslong as the modified base(s) maintains the same specificity for itsnatural complement through Watson-Crick base pairing (e.g., thepalindromic portion of the IMP is still self-complementary).

An IMP may be linear, may be circular or include circular portionsand/or may include a hairpin loop. In some embodiments, the IMPcomprises the following cyclic sequence (palindromic sequencesunderlined):

An IMP may be single stranded or double stranded DNA, as well as singleor double-stranded RNA or other modified polynucleotides. In someembodiments, the IMP comprises the following double-stranded sequences:

5′-TCGTCGAACGTTCGAGATGAT/5′-ATCATCTCGAACGTTCGACGA(SEQ ID NO: 27/SEQ ID NO: 29 duplex)  5′-TCG*TCG*AACG*TTCG*AG*ATG*AT/5′-ATCATCTCGAACGTTCGACGA (G* = 7-deaza-8-aza-dG, SEQID NO: 187/SEQ ID NO: 29 duplex) 5′-TCGTCGA*A*CGTTCGA*GA*TGA*T/5′-ATCATCTCGAACGTTCGACGA (A* = 2-amino-dA, SEQ ID NO:188/SEQ ID NO: 29 duplex)  5′-TCGTCGAA*CGT*TCGAGATGAT/5′-ATCATCTCGAACGTTCGACGA (A* = 2-amino-dA; T* = 2-thio-dT, SEQ ID NO: 189/SEQ ID NO: 29 duplex)5′-TCGTCGA*A*CGT*T*CGAGATGAT/5′- ATCATCTCGAACGTTCGACGA (A* =2-amino-dA; T* = 2- thio-dT, SEQ ID NO: 190/SEQ ID NO: 29 duplex).

An IMP may contain naturally-occurring or modified, non-naturallyoccurring bases, and may contain modified sugar, phosphate, and/ortermini. For example, in addition to phosphodiester linkages, phosphatemodifications include, but are not limited to, methyl phosphonate,phosphorothioate, phosphoramidate (bridging or non-bridging),phosphotriester and phosphorodithioate and may be used in anycombination. Other non-phosphate linkages may also be used. In someembodiments, polynucleotides of the present invention comprise onlyphosphorothioate backbones. In some embodiments, polynucleotides of thepresent invention comprise only phosphodiester backbones. In someembodiments, an IMP may comprise a combination of phosphate linkages inthe phosphate backbone such as a combination of phosphodiester andphosphorothioate linkages. For example, in some embodiments, the IMPcomprises the following sequences (“s” indicates phosphorothioatelinkages):

(SEQ ID NO: 62) 5′-TCGTCGAAACGTTTCGACAGT, all phosphorothioate linkages;(SEQ ID NO: 88) 5′-TCGTTCGAACGTTCGAACGA, all phosphodiester linkages;(SEQ ID NO: 89) 5′-TsCsGsTTCGAACGTTCGsAsAsCsGsA, phosphorothioate/phosphodiester chimera; (SEQ ID NO: 26)5′-GsGsTCGAACGTTCGAGsGsGsGsGsG, phosphorothioate/phosphodiester chimera; (SEQ ID NO: 33)5′-TsCsGsTCGAACGTTCGAGsGsGsGsGsG, phosphorothio-ate/phosphodiester chimera; (SEQ ID NO: 34)5′-TsCsGsTGCATCGATGCAGGsGsGsGsG, phosphorothioate/phosphodiester chimera.

Sugar modifications known in the field, such as 2′-alkoxy-RNA analogs,2′-amino-RNA analogs, 2′-fluoro-DNA, and 2′-alkoxy- or amino-RNA/DNAchimeras and others described herein, may also be made and combined withany phosphate modification. Examples of base modifications (discussedfurther below) include, but are not limited to, addition of anelectron-withdrawing moiety to C-5 and/or C-6 of a cytosine of the IMP(e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine,5-iodocytosine) and C-5 and/or C-6 of a uracil of the IMP (e.g.,5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil). See, forexample, International Patent Application No. WO 99/62923. As notedabove, use of a base modification in a palidromic sequence of an IMPshould not interfere with the self-complimentary ability of the basesinvolved for Watson-Crick base pairing. However, outside of apalindromic sequence, modified bases may be used without thisrestriction. For example, in some embodiments, the IMP comprises thefollowing sequences:

(SEQ ID NO: 21) 5′-uCGuCGAACGTTCGAGATG, u = 2′-O-methyl-uridine;(SEQ ID NO: 22) 5′-TcGTCGAACGTTCGAGATG, c = 2′-O-methyl-cytidine;(SEQ ID NO: 23) 5′-TCGTcGAACGTTCGAGATG, c = 2′-O-methyl-cytidine;(SEQ ID NO: 28) 5′-TBGTBGAABGTTBGAGATGAT, B = 5′-bromo-2′-deoxycytidine.

The IMP can be synthesized using techniques and nucleic acid synthesisequipment which are well known in the art including, but not limited to,enzymatic methods, chemical methods, and the degradation of largeroligonucleotide sequences. See, for example, Ausubel et al. (1987); andSambrook et al. (1989). When assembled enzymatically, the individualunits can be ligated, for example, with a ligase such as T4 DNA or RNAligase. U.S. Pat. No. 5,124,246. Oligonucleotide degradation can beaccomplished through the exposure of an oligonucleotide to a nuclease,as exemplified in U.S. Pat. No. 4,650,675.

The IMP can also be isolated using conventional polynucleotide isolationprocedures. Such procedures include, but are not limited to,hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences, antibody screening of expression libraries todetect shared structural features and synthesis of particular nativesequences by the polymerase chain reaction.

Circular immunomodulatory polynucleotide can be isolated, synthesizedthrough recombinant methods, or chemically synthesized. Where thecircular IMP is obtained through isolation or through recombinantmethods, the IMP will preferably be a plasmid. The chemical synthesis ofsmaller circular oligonucleotides can be performed using any methoddescribed in the literature. See, for instance, Gao et al. (1995)Nucleic Acids Res. 23:2025-2029; and Wang et al. (1994) Nucleic AcidsRes. 22:2326-2333.

Duplex (i.e., double stranded) and hairpin forms of most IMPs are indynamic equilibrium, with the hairpin form generally favored at lowpolynucleotide concentration and higher temperatures. Covalentinterstrand or intrastrand cross-links increases duplex or hairpinstability, respectively, towards thermal-, ionic-, pH-, andconcentration-induced conformational changes. Chemical cross-links canbe used to lock the polynucleotide into either the duplex or the hairpinform for physicochemical and biological characterization. Cross-linkedIMPs that are conformationally homogeneous and are “locked” in theirmost active form (either duplex or hairpin form) could potentially bemore active than their uncross-linked counterparts. Accordingly, someIMPs of the invention contain covalent interstrand and/or intrastrandcross-links.

A variety of ways to chemically cross-link duplex DNA are known in theart. Any cross-linking method may be used as long as the cross-linkedpolynucleotide product possesses the desired immunomodulatory activity.

One method, for example, results in a disulfide bridge between twoopposing thymidines at the terminus of the duplex or hairpin. For thiscross-linking method, the oligonucleotide(s) of interest is synthesizedwith a 5′-DMT-N3-(tBu-SS-ethyl)thymidine-3′-phosphoramidite (“T*”). Toform the disulfide bridge, the mixed disulfide bonds are reduced,oligonucleotide purified, the strands hybridized and the compoundair-oxidized to form the intrastrand cross-link in the case of a hairpinform or the interstrand cross-link in the case of a duplex form.Alternatively, the oligonucleotides may be hybridized first and thenreduced, purified and air-oxidized. Such methods and others aredescribed, for example, in Glick et al. (1991) J. Org. Chem.56:6746-6747, Glick et al. (1992) J. Am. Chem. Soc. 114:5447-5448,Goodwin et al. (1994) Tetrahedron Letters 35:1647-1650, Wang et al.(1995) J. Am. Chem. Soc. 117:2981-2991, Osborne et al. (1996) Bioorganic& Medicinal Chemistry Letters 6:2339-2342 and Osborne et al. (1996) J.Am. Chem. Soc. 118:11993-12003.

Examples of polynucleotide sequences in which a5′-DMT-N3-(tBu-SS-ethyl)thymidine-3′-phosphoramidite (“T*”) may beincorporated for the purpose of cross-linking include the following.Incorporation of the T* at the 3′ end of a SEQ ID NO:27 analog(5′-TCGTCGAACGTTCGAGATGAT*-3′, SEQ ID NO: 185) and at the 5′ end of aSEQ ID NO:29 analog (5′-T*TCATCTCGAACGTTCGACGA-3′, SEQ ID NO:186) wouldallow a cross-link in a duplex of the two strands at the 3′ end of theSEQ ID NO:27 analog. Incorporation of the T* at two locations in a SEQID NO:113 analog would allow two cross-links to form a duplex or asingle cross-link to hold a hairpin form. For example, folding of thesequence 5′-TCGT*AACGTTCGAACGTTCGAACGTTT*-3 (SEQ ID NO:227) into ahairpin structure and forming a cross-link at the substituted T residueswould result in a cross-linked polynucleotide with the followingsecondary structure.

                   A 5′-TCGT*AACGTTCGA    C     3′-T*TTGCAAGCT   G                   TSuch a hairpin structure or a duplex structure of the same sequencewould have a free 5′-TCG although constrained at two positions (the 3′end and 4 bases in from the 5′-end).

Another cross-linking method forms a disulfide bridge between offsetresidues in the duplex or hairpin structure. For this cross-linkingmethod, the oligonucleotide(s) of interest is synthesized withconvertible nucleosides (commercially available, for example, from GlenResearch). This method utilizes, for example, an A-A disulfide or a C-Adisulfide bridge and linkages through other bases are also possible. Toform the disulfide-modified polynucleotide, the polynucleotidecontaining the convertible nucleoside is reacted with cystamine (orother disulfide-containing amine). To form the disulfide bridge, themixed disulfide bonds are reduced, oligonucleotide purified, the strandshybridized and the compound air-oxidized to form the intrastrandcross-link in the case of a hairpin form or the interstrand cross-linkin the case of a duplex form. Alternatively, the oligonucleotides may behybridized first and then reduced, purified and air-oxidized. Suchmethods are described, for example, in Ferentz et al. (1991) J. Am.Chem. Soc. 113:4000-4002 and Ferentz et al. (1993) J. Am. Chem. Soc.115:9006-9014.

Examples of polynucleotide sequences in which offset N6-cystamine-2′-dA(A*) residues are used to crosslink a duplex include the following.Incorporation of the A* at the 3′ end of a the sequence5′-TCGTCGAACGTTCGAGA*TGAT-3′, SEQ ID NO:191 and at the 5′ end of itscomplement 5′-ATCA*TCTCGAACGTTCGACGA-3′, SEQ ID NO:192 would allow across-link in a duplex of the two strands at the 3′ end of the SEQ IDNO:191. Such modifications may also be used to cross-link hairpinstructures.

The techniques for making polynucleotides and modified polynucleotidesare known in the art. Naturally occurring DNA or RNA, containingphosphodiester linkages, is generally synthesized by sequentiallycoupling the appropriate nucleoside phosphoramidite to the 5′-hydroxygroup of the growing oligonucleotide attached to a solid support at the3′-end, followed by oxidation of the intermediate phosphite triester toa phosphate triester. Once the desired polynucleotide sequence has beensynthesized, the polynucleotide is removed from the support, thephosphate triester groups are deprotected to phosphate diesters and thenucleoside bases are deprotected using aqueous ammonia or other bases.See, for example, Beaucage (1993) “Oligodeoxyribonucleotide Synthesis”in Protocols for Oligonucleotides and Analogs, Synthesis and Properties(Agrawal, ed.) Humana Press, Totowa, N.J.; Warner et al. (1984) DNA3:401 and U.S. Pat. No. 4,458,066.

The IMP can also contain phosphate-modified polynucleotides, some ofwhich are known to stabilize the polynucleotide. Accordingly, someembodiments includes stabilized immunomodulatory polynucleotides.Synthesis of polynucleotides containing modified phosphate linkages ornon-phosphate linkages is also known in the art. For a review, seeMatteucci (1997) “Oligonucleotide Analogs: an Overview” inOligonucleotides as Therapeutic Agents, (D. J. Chadwick and G. Cardew,ed.) John Wiley and Sons, New York, N.Y. The phosphorous derivative (ormodified phosphate group) which can be attached to the sugar or sugaranalog moiety in the polynucleotides of the present invention can be amonophosphate, diphosphate, triphosphate, alkylphosphonate,phosphorothioate, phosphorodithioate, phosphoramidate or the like. Thepreparation of the above-noted phosphate analogs, and theirincorporation into nucleotides, modified nucleotides andoligonucleotides, per se, is also known and need not be described herein detail. Peyrottes et al. (1996) Nucleic Acids Res. 24:1841-1848;Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-2323; and Schultz etal. (1996) Nucleic Acids Res. 24:2966-2973. For example, synthesis ofphosphorothioate oligonucleotides is similar to that described above fornaturally occurring oligonucleotides except that the oxidation step isreplaced by a sulfurization step (Zon (1993) “OligonucleosidePhosphorothioates” in Protocols for Oligonucleotides and Analogs,Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190).Similarly the synthesis of other phosphate analogs, such asphosphotriester (Miller et al. (1971) JACS 93:6657-6665), non-bridgingphosphoramidates (Jager et al. (1988) Biochem. 27:7247-7246), N3′ to P5′phosphoramidiates (Nelson et al. (1997) JOC 62:7278-7287) andphosphorodithioates (U.S. Pat. No. 5,453,496) has also been described.Other non-phosphorous based modified oligonucleotides can also be used(Stirchak et al. (1989) Nucleic Acids Res. 17:6129-6141).Polynucleotides with phosphorothioate backbones can be more immunogenicthan those with phosphodiester backbones and appear to be more resistantto degradation after injection into the host. Braun et al. (1988) J.Immunol. 141:2084-2089; and Latimer et al. (1995) Mol. Immunol.32:1057-1064.

IMPs used in the invention can comprise one or more ribonucleotides(containing ribose as the only or principal sugar component),deoxyribonucleotides (containing deoxyribose as the principal sugarcomponent), or, as is known in the art, modified sugars or sugar analogscan be incorporated in the IMP. Thus, in addition to ribose anddeoxyribose, the sugar moiety can be pentose, deoxypentose, hexose,deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar “analog”cyclopentyl group. The sugar can be in pyranosyl or in a furanosyl form.In the IMP, the sugar moiety is preferably the furanoside of ribose,deoxyribose, arabinose or 2′-O-alkylribose, and the sugar can beattached to the respective heterocyclic bases either in α or β anomericconfiguration. Sugar modifications include, but are not limited to,2′-alkoxy-RNA analogs, 2′-amino-RNA analogs, 2′-fluoro-DNA, and2′-alkoxy- or amino-RNA/DNA chimeras. For example, a sugar modificationin the IMP includes, but is not limited to, 2′-O-methyl-uridine and2′-O-methyl-cytidine. The preparation of these sugars or sugar analogsand the respective “nucleosides” wherein such sugars or analogs areattached to a heterocyclic base (nucleic acid base) per se is known, andneed not be described here, except to the extent such preparation canpertain to any specific example. Sugar modifications may also be madeand combined with any phosphate modification in the preparation of anIMP.

The heterocyclic bases, or nucleic acid bases, which are incorporated inthe IMP can be the naturally-occurring principal purine and pyrimidinebases, (namely uracil, thymine, cytosine, adenine and guanine, asmentioned above), as well as naturally-occurring and syntheticmodifications of said principal bases. Thus, an IMP may include2′-deoxyuridine and/or 2-amino-2′-deoxyadenosine.

Those skilled in the art will recognize that a large number of“synthetic” non-natural nucleosides comprising various heterocyclicbases and various sugar moieties (and sugar analogs) are available inthe art, and that as long as other criteria of the present invention aresatisfied, the IMP can include one or several heterocyclic bases otherthan the principal five base components of naturally-occurring nucleicacids. Preferably, however, the heterocyclic base in the IMP includes,but is not limited to, uracil-5-yl, cytosin-5-yl, adenin-7-yl,adenin-8-yl, guanin-7-yl, guanin-8-yl,4-aminopyrrolo[2,3-d]pyrimidin-5-yl,2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl,2-amino-4-oxopyrrolo[2,3-d]pyrimidin-3-yl groups, where the purines areattached to the sugar moiety of the IMP via the 9-position, thepyrimidines via the 1-position, the pyrrolopyrimidines via the7-position and the pyrazolopyrimidines via the 1-position.

The IMP may comprise at least one modified base. As used herein, theterm “modified base” is synonymous with “base analog”, for example,“modified cytosine” is synonymous with “cytosine analog.” Similarly,“modified” nucleosides or nucleotides are herein defined as beingsynonymous with nucleoside or nucleotide “analogs.” Examples of basemodifications include, but are not limited to, addition of anelectron-withdrawing moiety to C-5 and/or C-6 of a cytosine of the IMP.Preferably, the electron-withdrawing moiety is a halogen. Such modifiedcytosines can include, but are not limited to, azacytosine,5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine,cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine,fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea,iodouracil, 5-nitrocytosine, uracil, and any other pyrimidine analog ormodified pyrimidine. Other examples of base modifications include, butare not limited to, addition of an electron-withdrawing moiety to C-5and/or C-6 of a uracil of the immunomodulatory polynucleotide.Preferably, the electron-withdrawing moiety is a halogen. Such modifieduracils can include, but are not limited to, 5-bromouracil,5-chlorouracil, 5-fluorouracil, 5-iodouracil.

Other examples of base modifications include the addition of one or morethiol groups to the base including, but not limited to, 2-amino-adenine,6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, and4-thio-uracil. Other examples of base modifications include, but are notlimited to, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine and5-hydroxycytosine. See, for example, Kandimalla et al. (2001) Bioorg.Med. Chem. 9:807-813. In some embodiments, the IMP comprises thefollowing sequences with modified bases (palindromic sequenceunderlined):

(SEQ ID NO: 193) 5′-TCXTCXAACXTTCXAGATGAT (X = 7-deaza-dG);(SEQ ID NO: 189) 5′-TCGTCGAA*CGT*TCGAGATGAT (A* = 2-amino-dA; T* =2-thio-dT); (SEQ ID NO: 190) 5′-TCGTCGA*A*CGT*T*CGAGATGAT (A* =2-amino-dA; T* = 2-thio-dT); (SEQ ID NO: 187)5′-TCG*TCG*AACG*TTCG*AG*ATG*AT (G* = 7-deaza-8- aza-dG);(SEQ ID NO: 194) 5′-TCG*AACG*TTCG*AACG*TTCG*AACG*TT (G* = 7-deaza-8-aza-dG); (SEQ ID NO: 195) 5′-TCGT*CGAACGT*T*CGAGAT*GAT* (T* =5-propynyl- dU); (SEQ ID NO: 196)5′-TCGAACGT*T*CGAACGT*T*CGAACGT*T* (T* = 5- propynyl-dU);(SEQ ID NO: 188) 5′-TCGTCGA*A*CGTTCGA*GA*TGA*T (A* = 2-amino-dA);(SEQ ID NO: 197) 5′-TCGA*A*CGTTCGA*A*CGTTCGA*A*CGTT(A* = 2-amino- dA).

As exemplified in Example 1, IMPs that maintain a duplex form at lowconcentration tend to be able to stimulate IFN-α production from humanPBMCs. Stabilizing duplex polynucleotide forms through cross-linking hasbeen described above. When in duplex form with their complementarysequence, certain modified bases also can increase the stability ofduplexes. For instance, 2-amino-dA (commercially available, for example,from Glen Research) forms 3 hydrogen bonds with T instead of 2 hydrogenbonds, as formed between dA and T. SEQ ID NO:188, an analog of SEQ IDNO:27, contains five 2-amino-dA bases in place of the five dA bases ofSEQ ID NO:27 and forms a stronger duplex with itself than SEQ ID NO:27(size exclusion chromatography data). Incorporation of these modifiedbases increases the Tm about 3° C. per modification. As demonstratedherein in Example 1, SEQ ID NO:188 also induced production of more IFN-αthan SEQ ID NO:27 when human PBMCs were treated with 0.8 μg/ml of IMP.Double-stranded SEQ ID NO:884 induced about three times the IFN-αproduction as single-stranded SEQ ID NO:188.

The preparation of base-modified nucleosides, and the synthesis ofmodified oligonucleotides using said base-modified nucleosides asprecursors, has been described, for example, in U.S. Pat. Nos.4,910,300, 4,948,882, and 5,093,232. These base-modified nucleosideshave been designed so that they can be incorporated by chemicalsynthesis into either terminal or internal positions of anoligonucleotide. Such base-modified nucleosides, present at eitherterminal or internal positions of an oligonucleotide, can serve as sitesfor attachment of a peptide or other antigen. Nucleosides modified intheir sugar moiety have also been described (including, but not limitedto, e.g., U.S. Pat. Nos. 4,849,513, 5,015,733, 5,118,800, 5,118,802) andcan be used similarly.

In some embodiments, an immunomodulatory polynucleotide is less thanabout any of the following lengths (in bases or base pairs): 10,000;5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175; 150;125; 100; 75; 60; 50; 40; 30; 25; 20; 15; 14; 13; 12; 11; 10. In someembodiments, an immunomodulatory polynucleotide is greater than aboutany of the following lengths (in bases or base pairs): 10; 11; 12; 13;14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300;350; 400; 500; 750; 1000; 2000; 5000; 7500; 10000; 20000; 50000.Alternately, the immunomodulatory polynucleotide can be any of a rangeof sizes having an upper limit of 10,000; 5,000; 2500; 2000; 1500; 1250;1000; 750; 500; 300; 250; 200; 175; 150; 125; 100; 75; 60; 50; 40; 30;25; 20; 15; 14; 13; 12; 11; 10 and an independently selected lower limitof 10; 11; 12; 13; 14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150;175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000; 7500, whereinthe lower limit is less than the upper limit In some embodiments, an IMPis preferably about 200 or less bases in length.

The invention also provides methods of making the immunomodulatorypolynucleotides described herein. The methods may be any of thosedescribed herein. For example, the method could be synthesizing the IMP(for example, using solid state synthesis) and may further comprise anypurification step(s). Methods of purification are known in the art.Other methods of preparation include combining an immunomodulatorypolynucleotide and an antigen.

Antigen

Any antigen may be co-administered with an immunomodulatorypolynucleotide and/or used in compositions comprising animmunomodulatory polynucleotide and antigen (and preparation of thesecompositions).

In some embodiments, the antigen is an allergen. Examples of recombinantallergens are provided in Table 1. Preparation of many allergens iswell-known in the art, including, but not limited to, preparation ofragweed pollen allergen Antigen E (Amb a I) (Rafnar et al. (1991) J.Biol. Chem. 266:1229-1236), grass allergen Lol p 1 (Tamborini et al.(1997) Eur. J. Biochem. 249:886-894), major dust mite allergens Der pIand Der PII (Chua et al. (1988) J. Exp. Med. 167:175-182; Chua et al.(1990) Int. Arch. Allergy Appl. Immunol. 91:124-129), domestic catallergen Fel d I (Rogers et al. (1993) Mol. Immunol. 30:559-568), whitebirch pollen Bet v1 (Breiteneder et al. (1989) EMBO J. 8:1935-1938),Japanese cedar allergens Cry j 1 and Cry j 2 (Kingetsu et al. (2000)Immunology 99:625-629), and protein antigens from other tree pollen(Elsayed et al. (1991) Scand. J. Clin. Lab. Invest. Suppl. 204:17-31).As indicated, allergens from trees are known, including allergens frombirch, juniper and Japanese cedar. Preparation of protein antigens fromgrass pollen for in vivo administration has been reported.

In some embodiments, the allergen is a food allergen, including, but notlimited to, peanut allergen, for example Ara h I (Stanley et al. (1996)Adv. Exp. Med. Biol. 409:213-216); walnut allergen, for example, Jug r I(Tueber et al. (1998) J. Allergy Clin. Immunol. 101:807-814); brazil nutallergen, for example, albumin (Pastorello et al. (1998) J. AllergyClin. Immunol. 102:1021-1027; shrimp allergen, for example, Pen a I(Reese et al. (1997) Int. Arch. Allergy Immunol. 113:240-242); eggallergen, for example, ovomucoid (Crooke et al. (1997) J. Immunol.159:2026-2032); milk allergen, for example, bovine β-lactoglobin (Selotal. (1999) Clin. Exp. Allergy 29:1055-1063); fish allergen, for example,parvalbumins (Van Do et al. (1999) Scand. J. Immunol. 50:619-625;Galland et al. (1998) J. Chromatogr. B. Biomed. Sci. Appl. 706:63-71).In some embodiments, the allergen is a latex allergen, including but notlimited to, Hey b 7 (Sowka et al. (1998) Eur. J. Biochem. 255:213-219).Table 1 shows an exemplary list of allergens that may be used.

TABLE 1 RECOMBINANT ALLERGENS Group Allergen Reference ANIMALS:CRUSTACEA Shrimp/lobster tropomyosin Leung et al. (1996) J. AllergyClin. Immunol. 98: 954-961 Pan s I Leung et al. (1998) Mol. Mar. Biol.Biotechnol. 7: 12-20 INSECTS Ant Sol i 2 (venom) Schmidt et al. JAllergy Clin Immunol., 1996, 98: 82-8 Bee Phospholipase A2 (PLA) Mulleret al. J Allergy Clin Immunol, 1995, 96: 395-402 Forster et al. JAllergy Clin Immunol, 1995, 95: 1229-35 Muller et al. Clin Exp Allergy,1997, 27: 915-20 Hyaluronidase (Hya) Soldatova et al. J Allergy ClinImmunol, 1998, 101: 691-8 Cockroach Bla g Bd9OK Helm et al. J AllergyClin Immunol, 1996, 98: 172-180 Bla g 4 (a calycin) Vailes et al. JAllergy Clin Immunol, 1998, 101: 274-280 Glutathione S- Arruda et al. JBiol Chem, 1997, 272: 20907-12 transferase Per a 3 Wu et al. MolImmunol, 1997, 34: 1-8 Dust mite Der p 2 (major allergen) Lynch et al. JAllergy Clin Immunol, 1998, 101: 562-4 Hakkaart et al. Clin Exp Allergy,1998, 28: 169-74 Hakkaart et al. Clin Exp Allergy, 1998, 28: 45-52Hakkaart et al. Int Arch Allergy Immunol, 1998, 115 (2): 150-6 Muelleret al. J Biol Chem, 1997, 272: 26893-8 Der p2 variant Smith et al. JAllergy Clin Immunol, 1998, 101: 423-5 Der f2 Yasue et al. Clin ExpImmunol, 1998, 113: 1-9 Yasue et al. Cell Immunol, 1997, 181: 30-7 Derp10 Asturias et al. Biochim Biophys Acta, 1998, 1397: 27-30 Tyr p 2Eriksson et al. Eur J Biochem, 1998 Hornet Antigen 5 aka Dol m VTomalski et al. Arch Insect Biochem Physiol, 1993, (venom) 22: 303-13Mosquito Aed a I (salivary Xu et al. Int Arch Allergy Immunol, 1998,115: 245-51 apyrase) Yellow jacket antigen 5, hyaluronidase King et al.J Allergy Clin Immunol, 1996, 98: 588-600 and phospholipase (venom)MAMMALS Cat Fel d I Slunt et al. J Allergy Clin Immunol, 1995, 95:1221-8 Hoffmann et al. (1997) J Allergy Clin Immunol 99: 227-32 HedlinCurr Opin Pediatr, 1995, 7: 676-82 Cow Bos d 2 (dander; a Zeiler et al.J Allergy Clin Immunol, 1997, 100: 721-7 lipocalin) Rautiainen et al.Biochem Bioph. Res Comm., 1998, 247: 746-50 β-lactoglobulin (BLG, Chatelet al. Mol Immunol, 1996, 33: 1113-8 major cow milk allergen) Lehrer etal. Crit Rev Food Sci Nutr, 1996, 36: 553-64 Dog Can f I and Can f 2,Konieczny et al. Immunology, 1997, 92: 577-86 salivary lipocalinsSpitzauer et al. J Allergy Clin Immunol, 1994, 93: 614-27 Vrtala et al.J Immunol, 1998, 160: 6137-44 Horse Equ c1 (major allergen, a Gregoireet al. J Biol Chem, 1996, 271: 32951-9 lipocalin) Mouse mouse urinaryprotein Konieczny et al. Immunology, 1997, 92: 577-86 (MUP) OTHERMAMMALIAN ALLERGENS Insulin Ganz et al. J Allergy Clin Immunol, 1990,86: 45-51 Grammer et al. J Lab Clin Med, 1987, 109: 141-6 Gonzalo et al.Allergy, 1998, 53: 106-7 Interferons interferon alpha 2c Detmar et al.Contact Dermatis, 1989, 20: 149-50 MOLLUSCS topomyosin Leung et al. JAllergy Clin Immunol, 1996, 98: 954-61 PLANT ALLERGENS: Barley Hor v 9Astwood et al. Adv Exp Med Biol, 1996, 409: 269-77 Birch pollenallergen, Bet v 4 Twardosz et al. Biochem Bioph. Res Comm., 1997, 239:197 rBet v 1 Bet v 2: Pauli et al. J Allergy Clin Immunol, 1996, 97:1100-9 (profilin) van Neerven et al. Clin Exp Allergy, 1998, 28: 423-33Jahn-Schmid et al. Immunotechnology, 1996, 2: 103-13 Breitwieser et al.Biotechniques, 1996, 21: 918-25 Fuchs et al. J Allergy Clin Immunol,1997, 100: 356-64 Brazil nut globulin Bartolome et al. AllergolImmunopathol, 1997, 25: 135-44 Cherry Pru a I (major allergen) Scheureret al. Mol Immunol, 1997, 34: 619-29 Corn Zml3 (pollen) Heiss et al.FEBS Lett, 1996, 381: 217-21 Lehrer et al. Int Arch Allergy Immunol,1997, 113: 122-4 Grass Phl p 1, Phl p 2, Phl p 5 Bufe et al. Am J RespirCrit Care Med, 1998, 157: 1269-76 (timothy grass pollen) Vrtala et al. JImmunol Jun 15, 1998, 160: 6137-44 Niederberger et al. J Allergy ClinImmun., 1998, 101: 258-64 Hol 1 5 velvet grass Schramm et al. Eur JBiochem, 1998, 252: 200-6 pollen Bluegrass allergen Zhang et al. JImmunol, 1993, 151: 791-9 Cyn d 7 Bermuda grass Smith et al. Int ArchAllergy Immunol, 1997, 114: 265-71 Cyn d 12 (a profilin) Asturias et al.Clin Exp Allergy, 1997, 27: 1307-13 Fuchs et al. J Allergy Clin Immunol,1997, 100: 356-64 Japanese Cedar Jun a 2 (Juniperus ashei) Yokoyama etal. Biochem. Biophys. Res. Commun., 2000, 275: 195-202 Cry j 1, Cry j 2Kingetsu et al. Immunology, 2000, 99: 625-629 (Cryptomeria japonica)Juniper Jun o 2 (pollen) Tinghino et al. J Allergy Chin Immunol, 1998,101: 772-7 Latex Hev b 7 Sowka et al. Eur J Biochem, 1998, 255: 213-9Fuchs et al. J Allergy Clin Immunol, 1997, 100: 356-64 Mercurialis Mer aI (profilin) Vallverdu et al. J Allergy Clin Immunol, 1998, 101: 363-70Mustard Sin a I (seed) Gonzalez de la Pena et al. Biochem Bioph. ResComm., (Yellow) 1993, 190: 648-53 Oilseed rape Bra r I pollen allergenSmith et al. Int Arch Allergy Immunol, 1997, 114: 265-71 Peanut Ara h IStanley et al. Adv Exp Med Biol, 1996, 409: 213-6 Burks et al. J ClinInvest, 1995, 96: 1715-21 Burks et al. Int Arch Allergy Immunol, 1995,107: 248-50 Poa pratensis Poa p9 Parronchi et al. Eur J Immunol, 1996,26: 697-703 Astwood et al. Adv Exp Med Biol, 1996, 409: 269-77 RagweedAmb a I Sun et al. Biotechnology Aug, 1995, 13: 779-86 Hirschwehr et al.J Allergy Clin Immunol, 1998, 101: 196-206 Casale et al. J Allergy ClinImmunol, 1997, 100: 110-21 Rye Lol p I Tamborini et al. Eur J Biochem,1997, 249: 886-94 Walnut Jug r I Teuber et al. J Allergy Clin Immun.,1998, 101: 807-14 Wheat allergen Fuchs et al. J Allergy Clin Immunol,1997, 100: 356-64 Donovan et al. Electrophoresis, 1993, 14: 917-22FUNGI: Aspergillus Asp f 1, Asp f 2, Asp f 3, Crameri et al. Mycoses,1998, 41 Suppl 1: 56-60 Asp f 4, rAsp f 6 Hemmann et al. Eur J Immunol,1998, 28: 1155-60 Banerjee et al. J Allergy Clin Immunol, 1997, 99:821-7 Crameri Int Arch Allergy Immunol, 1998, 115: 99-114 Crameri et al.Adv Exp Med Biol, 1996, 409: 111-6 Moser et al. J Allergy Clin Immunol,1994, 93: 1-11 Manganese superoxide Mayer et al. Int Arch AllergyImmunol, 1997, 113: 213-5 dismutase (MNSOD) Blomia allergen Caraballo etal. Adv Exp Med Biol, 1996, 409: 81-3 Penicillinium allergen Shen et al.Clin Exp Allergy, 1997, 27: 682-90 Psilocybe Psi c 2 Horner et al. IntArch Allergy Immunol, 1995, 107: 298-300

In some embodiments, the antigen is from an infectious agent, includingprotozoan, bacterial, fungal (including unicellular and multicellular),and viral infectious agents. Examples of suitable viral antigens aredescribed herein and are known in the art. Bacteria include Hemophilusinfluenza, Mycobacterium tuberculosis and Bordetella pertussis.Protozoan infectious agents include malarial plasmodia, Leishmaniaspecies, Trypanosoma species and Schistosoma species. Fungi includeCandida albicans.

In some embodiments, the antigen is a viral antigen. Viral polypeptideantigens include, but are not limited to, HIV proteins such as HIV gagproteins (including, but not limited to, membrane anchoring (MA)protein, core capsid (CA) protein and nucleocapsid (NC) protein), HIVpolymerase, influenza virus matrix (M) protein and influenza virusnucleocapsid (NP) protein, hepatitis B surface antigen (HBsAg),hepatitis B core protein (HBcAg), hepatitis e protein (HBeAg), hepatitisB DNA polymerase, hepatitis C antigens, and the like. Referencesdiscussing influenza vaccination include Scherle and Gerhard (1988)Proc. Natl. Acad. Sci. USA 85:4446-4450; Scherle and Gerhard (1986) J.Exp. Med. 164:1114-1128; Granoff et al. (1993) Vaccine 11:S46-51;Kodihalli et al. (1997) J. Virol. 71:3391-3396; Ahmeida et al. (1993)Vaccine 11:1302-1309; Chen et al. (1999) Vaccine 17:653-659; Govorkovaand Smirnov (1997) Acta Virol. (1997) 41:251-257; Koide et al. (1995)Vaccine 13:3-5; Mbawuike et al. (1994) Vaccine 12:1340-1348; Tamura etal. (1994) Vaccine 12:310-316; Tamura et al. (1992) Eur. J. Immunol.22:477-481; Hirabayashi et al. (1990) Vaccine 8:595-599. Other examplesof antigen polypeptides are group- or sub-group specific antigens, whichare known for a number of infectious agents, including, but not limitedto, adenovirus, herpes simplex virus, papilloma virus, respiratorysyncytial virus and poxviruses.

Many antigenic peptides and proteins are known, and available in theart; others can be identified using conventional techniques. Forimmunization against tumor formation or treatment of existing tumors,immunomodulatory peptides can include tumor cells (live or irradiated),tumor cell extracts, or protein subunits of tumor antigens such asHer-2/neu, Mart1, carcinoembryonic antigen (CEA), gangliosides, humanmilk fat globule (HMFG), mucin (MUC1), MAGE antigens, BAGE antigens,GAGE antigens, gp100, prostate specific antigen (PSA), and tyrosinase.Vaccines for immuno-based contraception can be formed by including spermproteins administered with IMP. Lea et al. (1996) Biochim. Biophys. Acta1307:263.

Attenuated and inactivated viruses are suitable for use herein as theantigen. Preparation of these viruses is well-known in the art and manyare commercially available (see, e.g., Physicians' Desk Reference (1998)52nd edition, Medical Economics Company, Inc.). For example, polio virusis available as IPOL® (Pasteur Merieux Connaught) and ORIMUNE® (LederleLaboratories), hepatitis A virus as VAQTA® (Merck), measles virus asATTENUVAX® (Merck), mumps virus as MUMPSVAX® (Merck) and rubella virusas MERUVAX®II (Merck). Additionally, attenuated and inactivated virusessuch as HIV-1, HIV-2, herpes simplex virus, hepatitis B virus,rotavirus, human and non-human papillomavirus and slow brain viruses canprovide peptide antigens.

In some embodiments, the antigen comprises a viral vector, such asvaccinia, adenovirus, and canary pox.

Antigens may be isolated from their source using purification techniquesknown in the art or, more conveniently, may be produced usingrecombinant methods.

Antigenic peptides can include purified native peptides, syntheticpeptides, recombinant proteins, crude protein extracts, attenuated orinactivated viruses, cells, micro-organisms, or fragments of suchpeptides. Immunomodulatory peptides can be native or synthesizedchemically or enzymatically. Any method of chemical synthesis known inthe art is suitable. Solution phase peptide synthesis can be used toconstruct peptides of moderate size or, for the chemical construction ofpeptides, solid phase synthesis can be employed. Atherton et al. (1981)Hoppe Seylers Z. Physiol. Chem. 362:833-839. Proteolytic enzymes canalso be utilized to couple amino acids to produce peptides. Kullmann(1987) Enzymatic Peptide Synthesis, CRC Press, Inc. Alternatively, thepeptide can be obtained by using the biochemical machinery of a cell, orby isolation from a biological source. Recombinant DNA techniques can beemployed for the production of peptides. Hames et al. (1987)Transcription and Translation: A Practical Approach, IRL Press. Peptidescan also be isolated using standard techniques such as affinitychromatography.

Preferably the antigens are peptides, lipids (e.g., sterols excludingcholesterol, fatty acids, and phospholipids), polysaccharides such asthose used in H. influenza vaccines, gangliosides and glycoproteins.These can be obtained through several methods known in the art,including isolation and synthesis using chemical and enzymatic methods.In certain cases, such as for many sterols, fatty acids andphospholipids, the antigenic portions of the molecules are commerciallyavailable.

Examples of viral antigens useful in the subject compositions andmethods using the compositions include, but are not limited to, HIVantigens. Such antigens include, but are not limited to, those antigensderived from HIV envelope glycoproteins including, but not limited to,gp160, gp120 and gp41. Numerous sequences for HIV genes and antigens areknown. For example, the Los Alamos National Laboratory HIV SequenceDatabase collects, curates and annotates HIV nucleotide and amino acidsequences. This database is accessible via the internet and in a yearlypublication, see Human Retroviruses and AIDS Compendium (for example,2000 edition).

Antigens derived from infectious agents may be obtained using methodsknown in the art, for example, from native viral or bacterial extracts,from cells infected with the infectious agent, from purifiedpolypeptides, from recombinantly produced polypeptides and/or assynthetic peptides.

IMP-Antigen

When used with antigen, IMP may be administered with antigen in a numberof ways. In some embodiments, an IMP and antigen may be administeredspatially proximate with respect to each other, or as an admixture(i.e., in solution). As described below, spatial proximation can beaccomplished in a number of ways, including conjugation (linkage),encapsidation, via affixation to a platform or adsorption onto asurface. Generally, and most preferably, an IMP and antigen areproximately associated at a distance effective to enhance the immuneresponse generated compared to the administration of the IMP and theantigen as an admixture.

In some embodiments, the IMP is conjugated with the antigen. The IMPportion can be coupled with the antigen portion of a conjugate in avariety of ways, including covalent and/or non-covalent interactions.

The link between the portions can be made at the 3′ or 5′ end of theIMP, or at a suitably modified base at an internal position in the IMP.If the antigen is a peptide and contains a suitable reactive group(e.g., an N-hydroxysuccinimide ester) it can be reacted directly withthe N⁴ amino group of cytosine residues. Depending on the number andlocation of cytosine residues in the IMP, specific coupling at one ormore residues can be achieved.

Alternatively, modified oligonucleosides, such as are known in the art,can be incorporated at either terminus, or at internal positions in theIMP. These can contain blocked functional groups which, when deblocked,are reactive with a variety of functional groups which can be presenton, or attached to, the antigen of interest.

Where the antigen is a peptide or polypeptide, this portion of theconjugate can be attached to the 3′-end of the IMP through solid supportchemistry. For example, the IMP portion can be added to a polypeptideportion that has been pre-synthesized on a support. Haralambidis et al.(1990a) Nucleic Acids Res. 18:493-499; and Haralambidis et al. (1990b)Nucleic Acids Res. 18:501-505. Alternatively, the IMP can be synthesizedsuch that it is connected to a solid support through a cleavable linkerextending from the 3′-end. Upon chemical cleavage of the IMP from thesupport, a terminal thiol group is left at the 3′-end of theoligonucleotide (Zuckermann et al. (1987) Nucleic Acids Res.15:5305-5321; and Corey et al. (1987) Science 238:1401-1403) or aterminal amino group is left at the 3′-end of the oligonucleotide(Nelson et al. (1989) Nucleic Acids Res. 17:1781-1794). Conjugation ofthe amino-modified IMP to amino groups of the peptide can be performedas described in Benoit et al. (1987) Neuromethods 6:43-72. Conjugationof the thiol-modified IMP to carboxyl groups of the peptide can beperformed as described in Sinah et al. (1991) Oligonucleotide Analogues:A Practical Approach, IRL Press. Coupling of an oligonucleotide carryingan appended maleimide to the thiol side chain of a cysteine residue of apeptide has also been described. Tung et al. (1991) Bioconjug. Chem.2:464-465.

The peptide or polypeptide portion of the conjugate can be attached tothe 5′-end of the IMP through an amine, thiol, or carboxyl group thathas been incorporated into the oligonucleotide during its synthesis.Preferably, while the oligonucleotide is fixed to the solid support, alinking group comprising a protected amine, thiol, or carboxyl at oneend, and a phosphoramidite at the other, is covalently attached to the5′-hydroxyl. Subsequent to deprotection, the amine, thiol, and carboxylfunctionalities can be used to covalently attach the oligonucleotide toa peptide. Benoit et al. (1987); and Sinah et al. (1991).

An IMP-antigen conjugate can also be formed through non-covalentinteractions, such as ionic bonds, hydrophobic interactions, hydrogenbonds and/or van der Waals attractions.

Non-covalently linked conjugates can include a non-covalent interactionsuch as a biotin-streptavidin complex. A biotinyl group can be attached,for example, to a modified base of an IMP. Roget et al. (1989) NucleicAcids Res. 17:7643-7651. Incorporation of a streptavidin moiety into thepeptide portion allows formation of a non-covalently bound complex ofthe streptavidin conjugated peptide and the biotinylatedoligonucleotide.

Non-covalent associations can also occur through ionic interactionsinvolving an IMP and residues within the antigen, such as charged aminoacids, or through the use of a linker portion comprising chargedresidues that can interact with both the oligonucleotide and theantigen. For example, non-covalent conjugation can occur between agenerally negatively-charged IMP and positively-charged amino acidresidues of a peptide, e.g., polylysine, polyarginine and polyhistidineresidues.

Non-covalent conjugation between IMP and antigens can occur through DNAbinding motifs of molecules that interact with DNA as their naturalligands. For example, such DNA binding motifs can be found intranscription factors and anti-DNA antibodies.

The linkage of the IMP to a lipid can be formed using standard methods.These methods include, but are not limited to, the synthesis ofoligonucleotide-phospholipid conjugates (Yanagawa et al. (1988) NucleicAcids Symp. Ser. 19:189-192), oligonucleotide-fatty acid conjugates(Grabarek et al. (1990) Anal. Biochem. 185:131-135; and Staros et al.(1986) Anal. Biochem. 156:220-222), and oligonucleotide-sterolconjugates. Boujrad et al. (1993) Proc. Natl. Acad. Sci. USA90:5728-5731.

The linkage of the oligonucleotide to an oligosaccharide can be formedusing standard known methods. These methods include, but are not limitedto, the synthesis of oligonucleotide-oligosaccharide conjugates, whereinthe oligosaccharide is a moiety of an immunoglobulin. O'Shannessy et al.(1985) J. Applied Biochem. 7:347-355.

The linkage of a circular IMP to a peptide or antigen can be formed inseveral ways. Where the circular IMP is synthesized using recombinant orchemical methods, a modified nucleoside is suitable. Ruth (1991) inOligonucleotides and Analogues: A Practical Approach, IRL Press.Standard linking technology can then be used to connect the circular IMPto the antigen or other peptide. Goodchild (1990) Bioconjug. Chem.1:165. Where the circular IMP is isolated, or synthesized usingrecombinant or chemical methods, the linkage can be formed by chemicallyactivating, or photoactivating, a reactive group (e.g. carbene, radical)that has been incorporated into the antigen or other peptide.

Additional methods for the attachment of peptides and other molecules tooligonucleotides can be found in U.S. Pat. No. 5,391,723; Kessler (1992)“Nonradioactive labeling methods for nucleic acids” in Kricka (ed.)Nonisotopic DNA Probe Techniques, Academic Press; and Geoghegan et al.(1992) Bioconjug. Chem. 3:138-146.

An IMP may be proximately associated with an antigen(s) in other ways.In some embodiments, an IMP and antigen are proximately associated byencapsulation. In other embodiments, an IMP and antigen are proximatelyassociated by linkage to a platform molecule. A “platform molecule”(also termed “platform”) is a molecule containing sites which allow forattachment of the IMP and antigen(s). In other embodiments, an IMP andantigen are proximately associated by adsorption onto a surface,preferably a carrier particle.

In some embodiments, the methods of the invention employ anencapsulating agent that can maintain the proximate association of theIMP and first antigen until the complex is available to the target (orcompositions comprising such encapsulating agents). Preferably, thecomposition comprising IMP, antigen and encapsulating agent is in theform of adjuvant oil-in-water emulsions, microparticles and/orliposomes. More preferably, adjuvant oil-in-water emulsions,microparticles and/or liposomes encapsulating an IMP-immunomodulatorymolecule are in the form of particles from about 0.04 μm to about 100 μmin size, preferably any of the following ranges: from about 0.1 μm toabout 20 μm; from about 0.15 μm to about 10 μm; from about 0.05 μm toabout 1.00 μm; from about 0.05 μm to about 0.5 μm.

Colloidal dispersion systems, such as microspheres, beads,macromolecular complexes, nanocapsules and lipid-based system, such asoil-in-water emulsions, micelles, mixed micelles and liposomes canprovide effective encapsulation of IMP-containing compositions.

The encapsulation composition further comprises any of a wide variety ofcomponents. These include, but are not limited to, alum, lipids,phospholipids, lipid membrane structures (LMS), polyethylene glycol(PEG) and other polymers, such as polypeptides, glycopeptides, andpolysaccharides.

Polypeptides suitable for encapsulation components include any known inthe art and include, but are not limited to, fatty acid bindingproteins. Modified polypeptides contain any of a variety ofmodifications, including, but not limited to glycosylation,phosphorylation, myristylation, sulfation and hydroxylation. As usedherein, a suitable polypeptide is one that will protect anIMP-containing composition to preserve the immunomodulatory activitythereof. Examples of binding proteins include, but are not limited to,albumins such as bovine serum albumin (BSA) and pea albumin.

Other suitable polymers can be any known in the art of pharmaceuticalsand include, but are not limited to, naturally-occurring polymers suchas dextrans, hydroxyethyl starch, and polysaccharides, and syntheticpolymers. Examples of naturally occurring polymers include proteins,glycopeptides, polysaccharides, dextran and lipids. The additionalpolymer can be a synthetic polymer. Examples of synthetic polymers whichare suitable for use in the present invention include, but are notlimited to, polyalkyl glycols (PAG) such as PEG, polyoxyethylatedpolyols (POP), such as polyoxyethylated glycerol (POG), polytrimethyleneglycol (PTG) polypropylene glycol (PPG), polyhydroxyethyl methacrylate,polyvinyl alcohol (PVA), polyacrylic acid, polyethyloxazoline,polyacrylamide, polyvinylpyrrolidone (PVP), polyamino acids,polyurethane and polyphosphazene. The synthetic polymers can also belinear or branched, substituted or unsubstituted, homopolymeric,co-polymers, or block co-polymers of two or more different syntheticmonomers.

The PEGs for use in encapsulation compositions of the present inventionare either purchased from chemical suppliers or synthesized usingtechniques known to those of skill in the art.

The term “LMS”, as used herein, means lamellar lipid particles whereinpolar head groups of a polar lipid are arranged to face an aqueous phaseof an interface to form membrane structures. Examples of the LMSsinclude liposomes, micelles, cochleates (i.e., generally cylindricalliposomes), microemulsions, unilamellar vesicles, multilamellarvesicles, and the like.

A preferred colloidal dispersion system of this invention is a liposome.In mice immunized with a liposome-encapsulated antigen, liposomesappeared to enhance a Th1-type immune response to the antigen. Aramakiet al. (1995) Vaccine 13:1809-1814. As used herein, a “liposome” or“lipid vesicle” is a small vesicle bounded by at least one and possiblymore than one bilayer lipid membrane. Liposomes are made artificiallyfrom phospholipids, glycolipids, lipids, steroids such as cholesterol,related molecules, or a combination thereof by any technique known inthe art, including but not limited to sonication, extrusion, or removalof detergent from lipid-detergent complexes. A liposome can alsooptionally comprise additional components, such as a tissue targetingcomponent. It is understood that a “lipid membrane” or “lipid bilayer”need not consist exclusively of lipids, but can additionally contain anysuitable other components, including, but not limited to, cholesteroland other steroids, lipid-soluble chemicals, proteins of any length, andother amphipathic molecules, providing the general structure of themembrane is a sheet of two hydrophilic surfaces sandwiching ahydrophobic core. For a general discussion of membrane structure, seeThe Encyclopedia of Molecular Biology by J. Kendrew (1994). For suitablelipids see e.g., Lasic (1993) “Liposomes: from Physics to Applications”Elsevier, Amsterdam.

Processes for preparing liposomes containing IMP-containing compositionsare known in the art. The lipid vesicles can be prepared by any suitabletechnique known in the art. Methods include, but are not limited to,microencapsulation, microfluidization, LLC method, ethanol injection,freon injection, the “bubble” method, detergent dialysis, hydration,sonication, and reverse-phase evaporation. Reviewed in Watwe et al.(1995) Curr. Sci. 68:715-724. Techniques may be combined in order toprovide vesicles with the most desirable attributes.

The invention encompasses use of LMSs containing tissue or cellulartargeting components. Such targeting components are components of a LMSthat enhance its accumulation at certain tissue or cellular sites inpreference to other tissue or cellular sites when administered to anintact animal, organ, or cell culture. A targeting component isgenerally accessible from outside the liposome, and is thereforepreferably either bound to the outer surface or inserted into the outerlipid bilayer. A targeting component can be inter alia a peptide, aregion of a larger peptide, an antibody specific for a cell surfacemolecule or marker, or antigen binding fragment thereof, a nucleic acid,a carbohydrate, a region of a complex carbohydrate, a special lipid, ora small molecule such as a drug, hormone, or hapten, attached to any ofthe aforementioned molecules. Antibodies with specificity toward celltype-specific cell surface markers are known in the art and are readilyprepared by methods known in the art.

The LMSs can be targeted to any cell type toward which a therapeutictreatment is to be directed, e.g., a cell type which can modulate and/orparticipate in an immune response. Such target cells and organs include,but are not limited to, APCs, such as macrophages, dendritic cells andlymphocytes, lymphatic structures, such as lymph nodes and the spleen,and nonlymphatic structures, particularly those in which dendritic cellsare found.

The LMS compositions of the present invention can additionally comprisesurfactants. Surfactants can be cationic, anionic, amphiphilic, ornonionic. A preferred class of surfactants are nonionic surfactants;particularly preferred are those that are water soluble.

In embodiments in which an IMP and antigen are proximately associated bylinkage to a platform molecule, the platform may be proteinaceous ornon-proteinaceous (i.e., organic). Examples of proteinaceous platformsinclude, but are not limited to, albumin, gammaglobulin, immunoglobulin(IgG) and ovalbumin Borel et al. (1990) Immunol. Methods 126:159-168;Dumas et al. (1995) Arch. Dematol. Res. 287:123-128; Borel et al. (1995)Int. Arch. Allergy Immunol. 107:264-267; Borel et al. (1996) Ann. N.Y.Acad. Sci. 778:80-87. A platform is multi-valent (i.e., contains morethan one binding, or linking, site) to accommodate binding to both anIMP and antigen. Accordingly, a platform may contain 2 or more, 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more binding or linking sites Other examples of polymericplatforms are dextran, polyacrylamide, ficoll, carboxymethylcellulose,polyvinyl alcohol, and poly D-glutamic acid/D-lysine.

The principles of using platform molecules are well understood in theart. Generally, a platform contains, or is derivatized to contain,appropriate binding sites for IMP and antigen. In addition, oralternatively, IMP and/or antigen is derivatized to provide appropriatelinkage groups. For example, a simple platform is a bi-functional linker(i.e., has two binding sites), such as a peptide. Further examples arediscussed below.

Platform molecules may be biologically stabilized, i.e., they exhibit anin vivo excretion half-life often of hours to days to months to confertherapeutic efficacy, and are preferably composed of a synthetic singlechain of defined composition. They generally have a molecular weight inthe range of about 200 to about 1,000,000, preferably any of thefollowing ranges: from about 200 to about 500,000; from about 200 toabout 200,000; from about 200 to about 50,000 (or less, such as 30,000).Examples of valency platform molecules are polymers (or are comprised ofpolymers) such as polyethylene glycol (PEG; preferably having amolecular weight of about 200 to about 8000), poly-D-lysine, polyvinylalcohol, polyvinylpyrrolidone, D-glutamic acid and D-lysine (in a ratioof 3:2). Other molecules that may be used are albumin and IgG.

Other platform molecules suitable for use within the present inventionare the chemically-defined, non-polymeric valency platform moleculesdisclosed in U.S. Pat. No. 5,552,391. Other homogeneouschemically-defined valency platform molecules suitable for use withinthe present invention are derivatized 2,2′-ethylenedioxydiethylamine(EDDA) and triethylene glycol (TEG).

Additional suitable valency platform molecules include, but are notlimited to, tetraminobenzene, heptaminobetacyclodextrin,tetraminopentaerythritol, 1,4,8,11-tetraazacyclotetradecane (Cyclam) and1,4,7,10-tetraazacyclododecane (Cyclen).

In general, these platforms are made by standard chemical synthesistechniques. PEG must be derivatized and made multivalent, which isaccomplished using standard techniques. Some substances suitable forconjugate synthesis, such as PEG, albumin, and IgG are availablecommercially.

Conjugation of an IMP and antigen to a platform molecule may be effectedin any number of ways, typically involving one or more crosslinkingagents and functional groups on the antigen and IMP platform andplatform molecule. Platforms and IMP and antigen must have appropriatelinking groups. Linking groups are added to platforms using standardsynthetic chemistry techniques. Linking groups may be added topolypeptide antigens and IMP using either standard solid phase synthetictechniques or recombinant techniques. Recombinant approaches may requirepost-translational modification in order to attach a linker, and suchmethods are known in the art.

As an example, polypeptides contain amino acid side chain moietiescontaining functional groups such as amino, carboxyl or sulfhydrylgroups that serve as sites for coupling the polypeptide to the platform.Residues that have such functional groups may be added to thepolypeptide if the polypeptide does not already contain these groups.Such residues may be incorporated by solid phase synthesis techniques orrecombinant techniques, both of which are well known in the peptidesynthesis arts. When the polypeptide has a carbohydrate side chain(s)(or if the antigen is a carbohydrate), functional amino, sulfhydryland/or aldehyde groups may be incorporated therein by conventionalchemistry. For instance, primary amino groups may be incorporated byreaction of the oxidized sugar with ethylenediamine in the presence ofsodium cyanoborohydride, sulfhydryls may be introduced by reaction ofcysteamine dihydrochloride followed by reduction with a standarddisulfide reducing agent, while aldehyde groups may be generatedfollowing periodate oxidation. In a similar fashion, the platformmolecule may also be derivatized to contain functional groups if it doesnot already possess appropriate functional groups.

Hydrophilic linkers of variable lengths are useful for connecting IMPand antigen to platform molecules. Suitable linkers include linearoligomers or polymers of ethylene glycol. Such linkers include linkerswith the formula R¹S(CH₂CH₂O)_(n)CH₂CH₂O(CH₂)_(m)CO₂R² wherein n=0-200,m=1 or 2, R¹=H or a protecting group such as trityl, R²=H or alkyl oraryl, e.g., 4-nitrophenyl ester. These linkers are useful in connectinga molecule containing a thiol reactive group such as haloaceyl,maleiamide, etc., via a thioether to a second molecule which contains anamino group via an amide bond. These linkers are flexible with regard tothe order of attachment, i.e., the thioether can be formed first orlast.

In embodiments in which an IMP and antigen are proximately associated byadsorption onto a surface, the surface may be in the form of a carrierparticle (for example, a nanoparticle) made with either an inorganic ororganic core. Examples of such nanoparticles include, but are notlimited to, nanocrystalline particles, nanoparticles made by thepolymerization of alkylcyanoacrylates and nanoparticles made by thepolymerization of methylidene malonate. Additional surfaces to which anIMP and antigen may be adsorbed include, but are not limited to,activated carbon particles and protein-ceramic nanoplates. Otherexamples of carrier particles are provided herein.

Adsorption of polynucleotides and polypeptides to a surface for thepurpose of delivery of the adsorbed molecules to cells is well known inthe art. See, for example, Douglas et al. (1987) Crit. Rev. Ther. Drug.Carrier Syst. 3:233-261; Hagiwara et al. (1987) In Vivo 1:241-252;Bousquet et al. (1999) Pharm. Res. 16:141-147; and Kossovsky et al.,U.S. Pat. No. 5,460,831. Preferably, the material comprising theadsorbent surface is biodegradable. Adsorption of an IMP and/or antigento a surface may occur through non-covalent interactions, includingionic and/or hydrophobic interactions.

In general, characteristics of carriers such as nanoparticles, such assurface charge, particle size and molecular weight, depend uponpolymerization conditions, monomer concentration and the presence ofstabilizers during the polymerization process (Douglas et al., 1987).The surface of carrier particles may be modified, for example, with asurface coating, to allow or enhance adsorption of the IMP and/orantigen. Carrier particles with adsorbed IMP and/or antigen may befurther coated with other substances. The addition of such othersubstances may, for example, prolong the half-life of the particles onceadministered to the subject and/or may target the particles to aspecific cell type or tissue, as described herein.

Nanocrystalline surfaces to which an IMP and antigen may be adsorbedhave been described (see, for example, U.S. Pat. No. 5,460,831).Nanocrystalline core particles (with diameters of 1 μm or less) arecoated with a surface energy modifying layer that promotes adsorption ofpolypeptides, polynucleotides and/or other pharmaceutical agents. Asdescribed in U.S. Pat. No. 5,460,831, for example, a core particle iscoated with a surface that promotes adsorption of an oligonucleotide andis subsequently coated with an antigen preparation, for example, in theform of a lipid-antigen mixture. Such nanoparticles are self-assemblingcomplexes of nanometer sized particles, typically on the order of 0.1μm, that carry an inner layer of IMP and an outer layer of antigen.

Another adsorbent surface are nanoparticles made by the polymerizationof alkylcyanoacrylates. Alkylcyanoacrylates can be polymerized inacidified aqueous media by a process of anionic polymerization.Depending on the polymerization conditions, the small particles tend tohave sizes in the range of 20 to 3000 nm, and it is possible to makenanoparticles specific surface characteristics and with specific surfacecharges (Douglas et al., 1987). For example, oligonucleotides may beadsorbed to polyisobutyl- and polyisohexlcyanoacrylate nanoparticles inthe presence of hydrophobic cations such as tetraphenylphosphoniumchloride or quaternary ammonium salts, such as cetyltrimethyl ammoniumbromide. Oligonucleotide adsorption on these nanoparticles appears to bemediated by the formation of ion pairs between negatively chargedphosphate groups of the nucleic acid chain and the hydrophobic cations.See, for example, Lambert et al. (1998) Biochimie 80:969-976, Chavany etal. (1994) Pharm. Res. 11:1370-1378; Chavany et al. (1992) Pharm. Res.9:441-449. Polypeptides may also be adsorbed to polyalkylcyanoacrylatenanoparticles. See, for example, Douglas et al., 1987; Schroeder et al.(1998) Peptides 19:777-780.

Another adsorbent surface are nanoparticles made by the polymerizationof methylidene malonate. For example, as described in Bousquet et al.,1999, polypeptides adsorbed to poly(methylidene malonate 2.1.2)nanoparticles appear to do so initially through electrostatic forcesfollowed by stabilization through hydrophobic forces.

IMP/MC Complexes

IMPs may be administered in the form of immunomodulatorypolynucleotide/microcarrier (IMP/MC) complexes. Accordingly, theinvention provides compositions comprising IMP/MC complexes.

Microcarriers useful in the invention are less than about 150, 120 or100 μm in size, more commonly less than about 50-60 μm in size,preferably less than about 10 μm in size, and are insoluble in purewater. Microcarriers used in the invention are preferably biodegradable,although nonbiodegradable microcarriers are acceptable. Microcarriersare commonly solid phase, such as “beads” or other particles, althoughliquid phase microcarriers such as oil in water emulsions comprising abiodegradable polymers or oils are also contemplated. A wide variety ofbiodegradable and nonbiodegradable materials acceptable for use asmicrocarriers are known in the art.

Microcarriers for use in the compositions or methods of the inventionare generally less than about 10 μm in size (e.g., have an averagediameter of less than about 10 μm, or at least about 97% of theparticles pass through a 10 μm screen filter), and include nanocarriers(i.e., carriers of less than about 1 μm size). Preferably, microcarriersare selected having sizes within an upper limit of about 9, 7, 5, 2, or1 μm or 900, 800, 700, 600, 500, 400, 300, 250, 200, or 100 nm and anindependently selected lower limit of about 4, 2, or 1 μm or about 800,600, 500, 400, 300, 250, 200, 150, 100, 50, 25, or 10 nm, where thelower limit is less than the upper limit. In some embodiments, themicrocarriers have a size of about 1.0-1.5 μm, about 1.0-2.0 μm or about0.9-1.6 μm. In certain preferred embodiments, the microcarriers have asize of about 10 nm to about 5 μm or about 25 nm to about 4.5 μm, about1 μm, about 1.2 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.8μm, about 2.0 μm, about 2.5 μm or about 4.5 μm. When the microcarriersare nanocarriers, preferred embodiments include nanocarriers of about 25to about 300 nm, 50 to about 200 nm, about 50 nm or about 200 nm.

Solid phase biodegradable microcarriers may be manufactured frombiodegradable polymers including, but not limited to: biodegradablepolyesters, such as poly(lactic acid), poly(glycolic acid), andcopolymers (including block copolymers) thereof, as well as blockcopolymers of poly(lactic acid) and poly(ethylene glycol);polyorthoesters such as polymers based on3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU);polyanhydrides such as poly(anhydride) polymers based on relativelyhydrophilic monomers such as sebacic acid; polyanhydride imides, such aspolyanhydride polymers based on sebacic acid-derived monomersincorporating amino acids (i.e., linked to sebacic acid by imide bondsthrough the amino-terminal nitrogen) such as glycine or alanine;polyanhydride esters; polyphosphazenes, especially poly(phosphazenes)which contain hydrolysis-sensitive ester groups which can catalyzedegradation of the polymer backbone through generation of carboxylicacid groups (Schacht et al., (1996) Biotechnol. Bioeng. 1996:102); andpolyamides such as poly(lactic acid-co-lysine).

A wide variety of nonbiodegradable materials suitable for manufacturingmicrocarriers are also known, including, but not limited to polystyrene,polypropylene, polyethylene, silica, ceramic, polyacrylamide, dextran,hydroxyapatite, latex, gold, and ferromagnetic or paramagneticmaterials. Certain embodiments exclude gold, latex, and/or magneticbeads. In certain embodiments, the microcarriers may be made of a firstmaterial (e.g., a magnetic material) encapsulated with a second material(e.g., polystyrene).

Solid phase microspheres are prepared using techniques known in the art.For example, they can be prepared by emulsion-solventextraction/evaporation technique. Generally, in this technique,biodegradable polymers such as polyanhydrates,poly(alkyl-α-cyanoacrylates) and poly(α-hydroxy esters), for example,poly(lactic acid), poly(glycolic acid), poly(D,L-lactic-co-glycolicacid) and poly(caprolactone), are dissolved in a suitable organicsolvent, such as methylene chloride, to constitute the dispersed phase(DP) of emulsion. DP is emulsified by high-speed homogenization intoexcess volume of aqueous continuous phase (CP) that contains a dissolvedsurfactant, for example, polyvinylalcohol (PVA) or polyvinylpirrolidone(PVP). Surfactant in CP is to ensure the formation of discrete andsuitably-sized emulsion droplet. The organic solvent is then extractedinto the CP and subsequently evaporated by raising the systemtemperature. The solid microparticles are then separated bycentrifugation or filtration, and dried, for example, by lyophilizationor application of vacuum, before storing at 4° C.

Physico-chemical characteristics such as mean size, size distributionand surface charge of dried microspheres may be determined. Sizecharacteristics are determined, for example, by dynamic light scatteringtechnique and the surface charge was determined by measuring the zetapotential.

Liquid phase microcarriers include liposomes, micelles, oil droplets andother lipid or oil-based particles which incorporate biodegradablepolymers or oils. In certain embodiments, the biodegradable polymer is asurfactant. In other embodiments, the liquid phase microcarriers arebiodegradable due to the inclusion of a biodegradable oil such assqualene or a vegetable oil. One preferred liquid phase microcarrier isoil droplets within an oil-in-water emulsion. Preferably, oil-in-wateremulsions used as microcarriers comprise biodegradable substituents suchas squalene.

IMP/MC complexes comprise an IMP bound to the surface of a microcarrier(i.e., the IMP is not encapsulated in the MC), and preferably comprisemultiple molecules of IMP bound to each microcarrier. In certainembodiments, a mixture of different IMPs may be complexed with amicrocarrier, such that the microcarrier is bound to more than one IMPspecies. The bond between the IMP and MC may be covalent ornon-covalent. As will be understood by one of skill in the art, the IMPmay be modified or derivatized and the composition of the microcarriermay be selected and/or modified to accommodate the desired type ofbinding desired for IMP/MC complex formation.

Covalently bonded IMP/MC complexes may be linked using any covalentcrosslinking technology known in the art. Typically, the IMP portionwill be modified, either to incorporate an additional moiety (e.g., afree amine, carboxyl or sulfhydryl group) or incorporate modified (e.g.,phosphorothioate) nucleotide bases to provide a site at which the IMPportion may be linked to the microcarrier. The link between the IMP andMC portions of the complex can be made at the 3′ or 5′ end of the IMP,or at a suitably modified base at an internal position in the IMP. Themicrocarrier is generally also modified to incorporate moieties throughwhich a covalent link may be formed, although functional groups normallypresent on the microcarrier may also be utilized. The IMP/MC is formedby incubating the IMP with a microcarrier under conditions which permitthe formation of a covalent complex (e.g., in the presence of acrosslinking agent or by use of an activated microcarrier comprising anactivated moiety which will form a covalent bond with the IMP).

A wide variety of crosslinking technologies are known in the art, andinclude crosslinkers reactive with amino, carboxyl and sulfhydrylgroups. As will be apparent to one of skill in the art, the selection ofa crosslinking agent and crosslinking protocol will depend on theconfiguration of the IMP and the microcarrier as well as the desiredfinal configuration of the IMP/MC complex. The crosslinker may be eitherhomobifunctional or heterobifunctional. When a homobifunctionalcrosslinker is used, the crosslinker exploits the same moiety on the IMPand MC (e.g., an aldehyde crosslinker may be used to covalently link anIMP and MC where both the IMP and MC comprise one or more free amines).Heterobifunctional crosslinkers utilize different moieties on the IMPand MC, (e.g., a maleimido-N-hydroxysuccinimide ester may be used tocovalently link a free sulfhydryl on the IMP and a free amine on theMC), and are preferred to minimize formation of inter-microcarrierbonds. In most cases, it is preferable to crosslink through a firstcrosslinking moiety on the microcarrier and a second crosslinking moietyon the IMP, where the second crosslinking moiety is not present on themicrocarrier. One preferred method of producing the IMP/MC complex is by‘activating’ the microcarrier by incubating with a heterobifunctionalcrosslinking agent, then forming the IMP/MC complex by incubating theIMP and activated MC under conditions appropriate for reaction. Thecrosslinker may incorporate a “spacer” arm between the reactivemoieties, or the two reactive moieties in the crosslinker may bedirectly linked.

In one preferred embodiment, the IMP portion comprises at least one freesulfhydryl (e.g., provided by a 5′-thiol modified base or linker) forcrosslinking to the microcarrier, while the microcarrier comprises freeamine groups. A heterobifunctional crosslinker reactive with these twogroups (e.g., a crosslinker comprising a maleimide group and aNHS-ester), such as succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate is used to activate theMC, then covalently crosslink the IMP to form the IMP/MC complex.

Non-covalent IMP/MC complexes may be linked by any non-covalent bindingor interaction, including ionic (electrostatic) bonds, hydrophobicinteractions, hydrogen bonds, van der Waals attractions, or acombination of two or more different interactions, as is normally thecase when a binding pair is to link the IMP and MC.

Preferred non-covalent IMP/MC complexes are typically complexed byhydrophobic or electrostatic (ionic) interactions, or a combinationthereof, (e.g., through base pairing between an IMP and a polynucleotidebound to an MC use of a binding pair). Due to the hydrophilic nature ofthe backbone of polynucleotides, IMP/MC complexes which rely onhydrophobic interactions to form the complex generally requiremodification of the IMP portion of the complex to incorporate a highlyhydrophobic moiety. Preferably, the hydrophobic moiety is biocompatible,nonimmunogenic, and is naturally occurring in the individual for whomthe composition is intended (e.g., is found in mammals, particularlyhumans). Examples of preferred hydrophobic moieties include lipids,steroids, sterols such as cholesterol, and terpenes. The method oflinking the hydrophobic moiety to the IMP will, of course, depend on theconfiguration of the IMP and the identity of the hydrophobic moiety. Thehydrophobic moiety may be added at any convenient site in the IMP,preferably at either the 5′ or 3′ end; in the case of addition of acholesterol moiety to an IMP, the cholesterol moiety is preferably addedto the 5′ end of the IMP, using conventional chemical reactions (see,for example, Godard et al. (1995) Eur. J. Biochem. 232:404-410).Preferably, microcarriers for use in IMP/MC complexes linked byhydrophobic bonding are made from hydrophobic materials, such as oildroplets or hydrophobic polymers, although hydrophilic materialsmodified to incorporate hydrophobic moieties may be utilized as well.When the microcarrier is a liposome or other liquid phase microcarriercomprising a lumen, the IMP/MC complex is formed by mixing the IMP andthe MC after preparation of the MC, in order to avoid encapsulation ofthe IMP during the MC preparation process.

Non-covalent IMP/MC complexes bound by electrostatic binding typicallyexploit the highly negative charge of the polynucleotide backbone.Accordingly, microcarriers for use in non-covalently bound IMP/MCcomplexes are generally positively charged (cationic) at physiologicalpH (e.g., about pH 6.8-7.4). The microcarrier may intrinsically possessa positive charge, but microcarriers made from compounds not normallypossessing a positive charge may be derivatized or otherwise modified tobecome positively charged (cationic). For example, the polymer used tomake the microcarrier may be derivatized to add positively chargedgroups, such as primary amines. Alternately, positively chargedcompounds may be incorporated in the formulation of the microcarrierduring manufacture (e.g., positively charged surfactants may be usedduring the manufacture of poly(lactic acid)/poly(glycolic acid)copolymers to confer a positive charge on the resulting microcarrierparticles).

As described herein, to prepare cationic microspheres, cationic lipidsor polymers, for example, 1,2-dioleoyl-1,2,3-trimethylammoniopropane(DOTAP), cetyltrimethylammonium bromide (CTAB) or polylysine, are addedeither to DP or CP, as per their solubility in these phases.

As described herein, IMP/MC complexes can be preformed by adsorptiononto cationic microspheres by incubation of polynucleotide and theparticles, preferably in an aqueous admixture. Such incubation may becarried out under any desired conditions, including ambient (room)temperature (e.g., approximately 20° C.) or under refrigeration (e.g.,4° C.). Because cationic microspheres and polynucleotides associaterelatively quickly, the incubation may be for any convenient timeperiod, such as 5, 10, 15 minutes or more, including overnight andlonger incubations. For example, IMPs can be adsorbed onto the cationicmicrospheres by overnight aqueous incubation of polynucleotide and theparticles at 4° C. However, because cationic microspheres andpolynucleotides spontaneously associate, the IMP/MC complex can beformed by simple co-administration of the polynucleotide and the MC.Microspheres may be characterized for size and surface charge before andafter polynucleotide association. Selected batches may then evaluatedfor activity against suitable controls in, for example, establishedhuman peripheral blood mononuclear cell (PBMC), as described herein, andmouse splenocyte assays. The formulations may also evaluated in suitableanimal models.

Non-covalent IMP/MC complexes linked by nucleotide base pairing may beproduced using conventional methodologies. Generally, base-paired IMP/MCcomplexes are produced using a microcarrier comprising a bound,preferably a covalently bound, polynucleotide (the “capturepolynucleotide”) that is at least partially complementary to the IMP.The segment of complementarity between the IMP and the capturenucleotide is preferably at least 6, 8, 10 or 15 contiguous base pairs,more preferably at least 20 contiguous base pairs. The capturenucleotide may be bound to the MC by any method known in the art, and ispreferably covalently bound to the IMP at the 5′ or 3′ end.

In other embodiments, a binding pair may be used to link the IMP and MCin an IMP/MC complex. The binding pair may be a receptor and ligand, anantibody and antigen (or epitope), or any other binding pair which bindsat high affinity (e.g., Kd less than about 10-8). One type of preferredbinding pair is biotin and streptavidin or biotin and avidin, which formvery tight complexes. When using a binding pair to mediate IMP/MCcomplex binding, the IMP is derivatized, typically by a covalentlinkage, with one member of the binding pair, and the MC is derivatizedwith the other member of the binding pair. Mixture of the twoderivatized compounds results in IMP/MC complex formation.

Many IMP/MC complex embodiments do not include an antigen, and certainembodiments exclude antigen(s) associated with the disease or disorderwhich is the object of the IMP/MC complex therapy. In furtherembodiments, the IMP is also bound to one or more antigen molecules.Antigen may be coupled with the IMP portion of an IMP/MC complex in avariety of ways, including covalent and/or non-covalent interactions, asdescribed, for example, in WO 98/16247. Alternately, the antigen may belinked to the microcarrier. The link between the antigen and the IMP inIMP/MC complexes comprising an antigen bound to the IMP can be made bytechniques described herein and known in the art, including, but notlimited to, direct covalent linkage, covalent conjugation via acrosslinker moiety (which may include a spacer arm), noncovalentconjugation via a specific binding pair (e.g., biotin and avidin), andnoncovalent conjugation via electrostatic or hydrophobic bonding.

IMP Complexes with Cationic Condensing Gent and Stabilizing Agent

IMPs may be administered as a composition comprising a cationiccondensing agent, an IMP, and a stabilizing agent (i.e., CIScomposition) for modulating an immune response in the recipient. See,U.S. Patent Application No. 60/402,968. In some embodiments, the CIScomposition may also comprise an antigen and/or a fatty acid.

The CIS compositions of the invention are typically in particulate form.As will be apparent to those of skill in the art, CIS particulatecompositions of the invention will consist of a population of particlesof different sizes. Due to this naturally arising variability, the“size” of the particles in the compositions of the invention may bedescribed in ranges or as a maximum or minimum diameter. Particles areconsidered to be a particular size if at least 95% of the particles (bymass) meet the specified dimension (e.g., if at least 97% of theparticles are less than 20 μm in diameter, then the composition isconsidered to consist of particles of less than 20 μm in diameter).Particle size may be measured by any convenient method known in the art,including filtration (e.g., use of a “depth” filter to capture particlesgreater than a cutoff size), dynamic light scattering, electronmicroscopy, including TEM (particularly in combination withfreeze-fracture processing) and SEM, and the like.

Preferably, the CIS compositions of the invention comprise particleswhich are less than about 50 μm in diameter, more preferably less thanabout 20 μm in diameter, although in some embodiments the particles willbe less than about 3, 2 or 1 μm in diameter. Preferred particle sizeranges include about 0.01 μm to 50 μm, 0.02 to 20 μm, 0.05 to 5 μm, and0.05 to 3 μm in diameter.

The components of the CIS compositions may be present in variousratios/quantities in the compositions, although it is contemplated thatthe amounts of the stabilizing agent(s) and optional components such asfatty acids and antigen will remain relatively invariant, withstabilizing agents generally ranging from about 0.1% to 0.5% (v/v),fatty acids ranging from about 0 to 0.5%, and antigen concentrationsranging from about 0.1 to about 100 μg/mL, preferably about 1 to about100 μg/mL, more preferably about 10 to 50 μg/mL. The amounts and ratiosof the IMP and the cationic condensing agent are subject to a greaterrange of variation in the compositions of the invention. The amount ofIMP will vary to a certain extent as a function of the molecular weightof the IMP, and generally ranges from about 50 μg/mL to about 2 mg/mL,preferably about 100 μg/mL to 1 mg/mL. The cationic condensing agent isgenerally present in excess (in terms of mass) over the IMP, generallyin ratios of about 1:2 (IMP:cationic condensing agent) to about 1:6,more preferably about 2:5 to 1:5.

Particle size in the CIS compositions is a function of a number ofvariables. The size distribution of particles in the compositions can bemodulated by altering the ratio of cationic condensing agent to IMP. Forexample, altering the ratio of cationic condensing agent to IMP in theexemplary +ISS/0.4% Tween 85/0.4% oleate/polymyxin B compositions canalter mean particle size from about 1.5 μm at cationic condensingagent:IMC=1 to about 45 μm at cationic condensing agent:IMP=10.

In certain embodiments, the CIS compositions comprise a cationiccondensing agent, an IMP and a stabilizing agent that is a nonionicdetergent. In other embodiments, the compositions comprise a membranedisrupting cationic lipopeptide (preferably a polymyxin, more preferablypolymyxin B), an IMP and a stabilizing agent. In some embodiments thestabilizing agent is not a serum protein (particularly not a bovineserum protein). An exemplary composition of this class of embodimentsutilizes a polyoxyethylene ether detergent such as Tween 80 or Tween 85as the stabilizing agent, with oleate as an optional additionalstabilizing agent.

In some embodiments, CIS compositions comprise immunomodulatoryparticles, wherein the particles are made by the process of combining acationic condensing agent, an IMP and a stabilizing agent that is anonionic detergent. In other embodiments, compositions of the inventioncomprise immunomodulatory particles, wherein the particles are made bythe process of combining a membrane disrupting cationic lipopeptide(preferably a polymyxin, more preferably polymyxin B), an IMP and astabilizing agent. In some embodiments the stabilizing agent is not aserum protein (particularly not a bovine serum protein).

In some embodiments, CIS compositions comprise immunomodulatoryparticles, wherein the particles are formed by the process of combiningan IMP and a stabilizing agent that is a nonionic detergent, therebyforming an IMP/stabilizing agent mixture, and combining a cationiccondensing agent with the IMP/stabilizing agent mixture. In otherembodiments, compositions of the invention comprise immunomodulatoryparticles, wherein the particles are formed by the process of combiningan IMP and a stabilizing agent, thereby forming an IMP/stabilizing agentmixture, and combining a membrane disrupting cationic lipopeptide(preferably a polymyxin, more preferably polymyxin B) with theIMP/stabilizing agent mixture. In some embodiments the stabilizing agentis not a serum protein (particularly not a bovine serum protein).

In some embodiments, CIS compositions comprise immunomodulatoryparticles, wherein the particles comprise a cationic condensing agent,an IMP and a stabilizing agent that is a nonionic detergent. In otherembodiments, compositions of the invention comprise immunomodulatoryparticles, wherein the particles comprise a membrane disrupting cationiclipopeptide (preferably a polymyxin, more preferably polymyxin B), anIMP and a stabilizing agent. In some embodiments the stabilizing agentis not a serum protein (particularly not a bovine serum protein).

Cationic condensing agents useful in the CIS compositions and methods ofusing the CIS compositions are molecules which are positively charged atphysiological pH (i.e., pH of about 7.0 to about 7.5). Preferably,cationic condensing agents used in the instant invention are notzwitterionic and are polycationic, that is, having more than onepositive charge per molecule. Cationic condensing agents useful in theinstant invention include hydrophilic or amphipathic polycations.

Preferred cationic condensing agents include: (a) membrane disruptingcationic lipopeptides including, but not limited to polymyxins includingpolymyxin A, polymyxin B (including polymyxin B₁ and polymyxin B₂),polymyxin C, polymyxin D, polymyxin E (also known as colistin),polymyxin K, polymyxin M, polymyxin P, polymyxin S and polymyxin T,circulins including circulin A, circulin B, circulin C, circulin D,circulin E and circulin F, octapeptin, amphotericins includingamphotericin B, and acylated peptides including octanoyl-KFFKFFKFF (SEQID NO:182) and acyl KALA (octanoyl-WEAKLAKALAKALAKHLAKALAKALEACEA (SEQID NO:183); (b) membrane disrupting cationic peptides including, but notlimited to polymyxin B nonapeptide, cecropins including cecropin A,cecropin B and cecropin P1, KFFKFFKFF (SEQ ID NO:182) and KALA(WEAKLAKALAKALAKHLAKALAKALKACEA) (SEQ ID NO:184); (c) single chaincationic surfactants including, but not limited tocetyltrimethylammonium bromide (CTAB), benzyl-dimethyl-ammonium bromide(BDAB), CpyrB (cetyl-pyridinium bromide), CimB (cetyl imidazoliumbromide), and polycationic polymers, including, but not limited to,poly-L-lysine (PLL) and polyethyleneimine (PEI). In certain embodiments,the cationic condensing agent is a membrane disrupting cationiclipopeptide, preferably a polymyxin, more preferably polymyxin B. Insome embodiments, cationic condensing agents may exclude fatty acidesters (i.e., lipids) and double chain cationic surfactants.

Stabilizing agents useful in the CIS compositions and methods of usingthe CIS compositions include those which are suspendable in water andreduce the surface tension of water, although stabilizing agents whichare water soluble and/or completely miscible in water are preferred. Anumber of classes of stabilizing agents are useful in the compositionsand methods of the invention, including proteins (preferably hydrophilicproteins), nonionic detergents, polymeric surfactants (e.g., polyvinylalcohol and polyvinyl pyrrolidone), cationic detergents, anionicdetergents and fatty acids, although in certain embodiments, serumproteins (particularly bovine serum proteins), fatty acids, and/or ionicdetergents may be excluded from the definition of stabilizing agents.

Any protein may be used as a stabilizing agent in accordance with theinvention. In some embodiments, the stabilizing agent is a protein whichis not intended as an antigen (see discussion below); in theseembodiments, it is preferred that the protein be derived from the samespecies as the intended recipient of the composition (e.g., if thecomposition is intended for use in humans, then it is preferred that theprotein used as the stabilizing agent be a human protein). Serum albuminis an exemplary protein useful as a stabilizing agent in suchembodiments. In other embodiments, an antigen is utilizing as thestabilizing agent, in which case the antigen need not be, and in generalis preferably not, species matched with the intended recipient. Antigensuseful in the compositions and methods of the invention are disclosedbelow.

Nonionic detergents useful in the CIS compositions and methods of usingthe CIS compositions include glucamides such as decyldimethylphosphineoxide (APO-10) and dimethyldodecylphosphine oxide (APO-12),octanoyl-N-methylglucamide (MEGA-8), nonanoyl-N-methylglucamide (MEGA-9)and decanoyl-N-methyl glucamide (MEGA-10), polyoxyethylene etherdetergents including polyoxyethylene(10) dodecyl ester (Genapol C100),polyoxyethylene(4) lauryl ether (BRIJ® 30), polyoxyethylene(9) laurylether (LUBROL® PX) polyoxyethylene(23) lauryl ether (BRIJ® 35),polyoxyethylene(2) cetyl ether (BRIJ® 52), polyoxyethylene(10) cetylether (BRIJ® 56), polyoxyethylene(20) cetyl ether (BRIJ® 58),polyoxyethylene(2) stearyl ether (BRIJ® 72), polyoxyethylene(10) stearylether (BRIJ® 76), polyoxyethylene(20) stearyl ether (BRIJ® 78),polyoxyethylene(100) stearyl ether (BRIJ® 700), polyoxyethylene(2) oleylether (BRIJ® 92), polyoxyethylene(10) oleyl ether (BRIJ® 97),polyoxyethylene(20) oleyl ether (BRIJ® 98),isotridecylpoly(ethyleneglycolether)₈ (Genapol 80), PLURONIC® F-68,PLURONIC® F-127, dodecylpoly(ethyleneglycolether)₉ (Thesit)polyoxyethylene(10) isooctylphenyl ether (TRITON® X-100),polyoxyethylene(8) isooctylphenyl ether (TRITON® X-114), polyethyleneglycol sorbitan monolaurate (TWEEN® 20), polyoxyethylenesorbitanmonopalmitate (TWEEN® 40), polyethylene glycol sorbitan monostearate(TWEEN® 60), polyoxyethylenesorbitan tristearate (TWEEN® 65),polyethylene glycol sorbitan monooleate (TWEEN® 80), polyoxyethylene(20)sorbitan trioleate (TWEEN® 85), poloxamer 188, andpolyethyleneglycol-p-isooctylphenyl ether (Nonidet NP40), alkylmaltoside detergents including cyclohexyl-n-ethyl-β-D-maltoside,cyclohexyl-n-hexyl-β-D-maltoside, and cyclohexyl-n-methyl-β-D-maltoside,n-decanoylsucrose, glucopyranosides including methyl6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside (HECAMEG) and alkylglucopyranosides such as n-decyl-β-D-glucopyranoside,n-heptyl-β-D-glucopyranoside, n-dodecyl-β-D-glucopyranoside,n-nonyl-β-D-glucopyranoside, n-octyl-α-D-glucopyranoside, andn-octyl-β-D-glucopyranoside, alkyl thioglucopyranosides includingn-heptyl-β-D-thioglucopyranoside, alkyl maltopyranosides includingn-decyl-β-D-maltopyranoside and n-octyl-β-D-maltopyranoside,n-decyl-β-D-thiomaltoside, digitonin, n-dodecanoyl sucrose,n-dodecyl-β-D-maltoside, heptane 1,2,3-triol,n-octanoyl-β-D-glucosylamine (NOGA), n-octanoyl sucrose, poloxamers(polyoxyethylene/polyoxypropylene block copolymers) such as poloxamer188 and poloxamer 407, and sulfobetaines including SB-10, SB-12, andSB-14 and n-undecyl-β-D-maltoside. Preferred stablizing agents includepolyoxyethylene ether detergents, particularly polyethylene glycolsorbitan monooleate and polyoxyethylene(20) sorbitan trioleate.

Anionic detergents useful in the CIS compositions and methods of usingthe CIS compositions include caprylic acid and salts thereof,chenodeoxycholic acid and salts thereof, cholic acid and salts thereof,decanesulfonic acid and salts thereof, deoxycholic acid and saltsthereof, glycodeoxycholic acid and salts thereof, lauroylsarcosine andsalts thereof, n-dodecyl sulfate and salts thereof (including sodium andlithium salts), taurochenodeoxycholic acid and salts thereof,taurocholic acid and salts thereof, taurodehydrocholic acid and saltsthereof, taurodeoxycholic acid and salts thereof, taurolithocholic acidand salts thereof, and tauroursodeoxycholic acid and salts thereof.

Cationic detergents include cetylpyridinium and salts thereof,cetyltrimethylamonia and salts thereof including cetyltrimethylammoniumbromide (CTAB), dodecyltrimethylammonia and salts thereof includingdedecyltrimethylammonium bromide, alklylammonium imidazolines,quaternary imidazolines, and tetradecyltrimtheylammonia and saltsthereof including tetradecyltrimtheylammonium bromide.

Detergents selected for use as stablizing agents are preferably thosethat are considered oil/water emulsifying detergents. Oil/wateremulsifying detergents are known in the art, and are generallycharacterized by a hydrophobic/lipophilic balance (HLB) value of about 8to about 18. Preferably, detergents incorporated into the particulatecompositions have HLB values of about 10 to about 16, more preferablyabout 11 to about 15 (e.g., polyethylene glycol sorbitan monooleate,HLB=15.4; polyoxyethylene(10) isooctylphenyl ether, HLB=13.5;polyoxyethylene(20) sorbitan trioleate HLB=11).

In certain embodiments, the CIS compositions may also include one ormore fatty acids, or a salt thereof, as an additional component. Inthose embodiments employing a fatty acid as the stablizing agentcomponent and a fatty acid as an additional component of thecomposition, the fatty acid utilized as the stablizing agent will bedifferent than the fatty acid used as the ‘additional’ component. Fattyacids useful in the CIS compositions of the invention may range in sizefrom four to 30 carbon atoms, and may be unsaturated (e.g., stearicacid), monounsaturated (e.g., oleic acid), or polyunsaturated (e.g.,linoleic acid), although monounsaturated and polyunsaturated fatty acidsare generally preferred.

In some embodiments, the CIS compositions will incorporate a fatty acidhaving a carbon chain length of at least about 4, 5, 6, 8, 10, 15, 18,or 20 carbon atoms and less than about 30, 25, 20, 19, 15 or 10 carbonatoms. Accordingly, in some embodiments the fatty acids utilized in theinvention may have carbon chains with a length in the range of about 4to 30, 5 to 25, 10 to 20, or 15 to 20 carbon atoms.

Fatty acids useful in the CIS compositions include, but are not limitedto, arachidonic acid, decanoic acid, docosanoic acid, docosahexanoicacid eicosanoic acid, heneicosanoic acid, heptadecanoic acid, heptanoicacid, hexanoic acid, lauric acid, linoleic acid, linolenic acid,myristic acid, nonadecanoic acid, nonanoic acid, octanoic acid, oleicacid, palmitic acid, pentadecanoic acid, stearic acid, tetracosanoicacid, tricosanoic acid, tridecanoic acid, and undecanoic acid. Preferredfatty acids for use in the CIS compositions include oleic acidpalmitoleic acid, and linoleic acid.

In certain embodiments of the invention, an antigen is incorporated intothe CIS composition or administered in combination with a CIScomposition. Those CIS compositions incorporating an antigen mayincorporate the antigen into the particulate composition itself, or bedissolved or suspended in the solution in which the particulatecomposition is suspended. Any antigen may be incorporated into orco-administered with a CIS composition of the invention.

METHODS OF THE INVENTION

As described herein, IMPs of the invention may particularly stimulateproduction of IL-6, TNF-α, IFN-γ and of type I interferons, includingIFN-α and IFN-ω, stimulate B cell proliferation and/or activateplasmacytoid dendritic cells to differentiate. The IMPs of the inventionmay also stimulate production of other cytokines, chemokines andactivation-associated proteins including, but not limited to, IP-10(interferon induced protein 10 kDa), MCP-1 (monocyte chemoattractantprotein 1), MCP-2, MCP-3, MIG, MIP-3b, CD80, CD86, CD40, CD54 and MHCclass II. The IMPs of the invention may also stimulate expression ofIFN-α-inducible genes including, but not limited to 2,5-oligoadenylatesynthatse (2,5-OAS), interferon-stimulating gene-54K (ISG-54K) andguanylate-binding protein-1 (GBP-1). The immunomodulatorypolynucleotides of the invention also may provide a signal that retardsplasmacytoid dendritic cell apoptosis. The immunomodulatorypolynucleotides of the invention also may stimulate natural killer (NK)cell lytic activity. Accordingly, the IMPs of the invention areparticularly effective in modulating an immune response in anindividual.

The invention provides methods of modulating an immune response in anindividual, preferably a mammal, more preferably a human, comprisingadministering to the individual an IMP as described herein.Immunomodulation may include stimulating a Th1-type immune responseand/or inhibiting or reducing a Th2-type immune response. The IMP isadministered in an amount sufficient to modulate an immune response. Asdescribed herein, modulation of an immune response may be humoral and/orcellular, and is measured using standard techniques in the art and asdescribed herein.

For example, the modulation of an immune response of an animal orpopulation of cells, e.g., mammalian, optionally human, blood cells(e.g., PBMCs, lymphocytes, dendritic cells), bronchial alveolar lavagecells, or other cells or cell populations containing ISS-responsivecells, is accomplished by contacting the cells with an IMP orIMP-containing composition described herein (e.g., a compositioncontaining an IMP, IMP and an antigen, an IMP-antigen conjugate, anIMP/microcarrier complex, etc.). The modulation can be accomplished byany form of contacting, including without limitation, co-incubation ofcells and IMP in vitro, application of the IMP to skin of a mammal(e.g., of an experimental animal), and parenteral administration.

An immune response in animals or cell populations can be detected in anynumber of ways, including increased expression of one or more of IFN-γ,IFN-α, IL-2, IL-12, TNF-α, IL-6, IL-4, IL-5, IP-10, ISG-54K, MCP-1, or achange in gene expression profile characteristics of immune stimulationas well as responses such as B cell proliferation and dendritic cellmaturation. The ability to stimulate an immune response in a cellpopulation has a number of uses, e.g., in an assay system forimmunosuppressive agents.

A number of individuals are suitable for receiving the immunomodulatorypolynucleotide(s) described herein. Preferably, but not necessarily, theindividual is human.

In certain embodiments, the individual suffers from a disorderassociated with a Th2-type immune response, such as allergies orallergy-induced asthma. Administration of an IMP results inimmunomodulation, increasing levels of one or more Th1-type responseassociated cytokines, which may result in a reduction of the Th2-typeresponse features associated with the individual's response to theallergen. Immunomodulation of individuals with Th2-type responseassociated disorders results in a reduction or improvement in one ormore of the symptoms of the disorder. Where the disorder is allergy orallergy-induced asthma, improvement in one or more of the symptomsincludes a reduction one or more of the following: rhinitis, allergicconjunctivitis, circulating levels of IgE, circulating levels ofhistamine and/or requirement for ‘rescue’ inhaler therapy (e.g., inhaledalbuterol administered by metered dose inhaler or nebulizer).

In further embodiments, the individual subject to the immunomodulatorytherapy of the invention is an individual receiving a vaccine. Thevaccine may be a prophylactic vaccine or a therapeutic vaccine. Aprophylactic vaccine comprises one or more epitopes associated with adisorder for which the individual may be at risk (e.g., M. tuberculosisantigens as a vaccine for prevention of tuberculosis). Therapeuticvaccines comprise one or more epitopes associated with a particulardisorder affecting the individual, such as M. tuberculosis or M. bovissurface antigens in tuberculosis patients, antigens to which theindividual is allergic (i.e., allergy desensitization therapy) inindividuals subject to allergies, tumor cells from an individual withcancer (e.g., as described in U.S. Pat. No. 5,484,596), or tumorassociated antigens in cancer patients.

The IMP may be given in conjunction with the vaccine (e.g., in the sameinjection or a contemporaneous, but separate, injection) or the IMP maybe administered separately (e.g., at least 12 hours before or afteradministration of the vaccine). In certain embodiments, the antigen(s)of the vaccine is part of the IMP, by either covalent or non-covalentlinkage to the IMP. In other embodiments, the IMP may be administeredalone as a prophylactic vaccine to increase resistance to infection by awide range of bacterial or viral pathogens, including natural orgenetically modified organisms employed as agents of biological warfareor terrorism. Administration of immunomodulatory polynucleotide therapyto an individual receiving a vaccine results in an immune response tothe vaccine that is shifted towards a Th1-type response as compared toindividuals which receive vaccine without IMP. Shifting towards aTh1-type response may be recognized by a delayed-type hypersensitivity(DTH) response to the antigen(s) in the vaccine, increased IFN-γ andother Th1-type response associated cytokines, production of CTLsspecific for the antigen(s) of the vaccine, low or reduced levels of IgEspecific for the antigen(s) of the vaccine, a reduction inTh2-associated antibodies specific for the antigen(s) of the vaccine,and/or an increase in Th1-associated antibodies specific for theantigen(s) of the vaccine. In the case of therapeutic vaccines,administration of IMP and vaccine results in amelioration of one or moresymptoms of the disorder which the vaccine is intended to treat. As willbe apparent to one of skill in the art, the exact symptom(s) and mannerof their improvement will depend on the disorder sought to be treated.For example, where the therapeutic vaccine is for tuberculosis, IMPtreatment with vaccine results in reduced coughing, pleural or chestwall pain, fever, and/or other symptoms known in the art. Where thevaccine is an allergen used in allergy desensitization therapy, thetreatment results in a reduction in the symptoms of allergy (e.g.,reduction in rhinitis, allergic conjunctivitis, circulating levels ofIgE, and/or circulating levels of histamine).

Other embodiments of the invention relate to immunomodulatory therapy ofindividuals having a pre-existing disease or disorder, such as cancer oran infectious disease. Cancer is an attractive target forimmunomodulation because most cancers express tumor-associated and/ortumor specific antigens which are not found on other cells in the body.Stimulation of a Th1-type response against tumor cells results in directand/or bystander killing of tumor cells by the immune system, leading toa reduction in cancer cells and/or a reduction in symptom(s).Administration of an IMP to an individual having cancer results instimulation of a Th1-type immune response against the tumor cells. Suchan immune response can kill tumor cells, either by direct action ofcellular immune system cells (e.g., CTLs, NK cells) or components of thehumoral immune system, or by bystander effects on cells proximal tocells targeted by the immune system. See, for example, Cho et al. (2000)Nat. Biotechnol. 18:509-514. In the cancer context, administration ofIMPs may further comprise administration of one or more additionaltherapeutic agents such as, for example, anti-tumor antibodies,chemotherapy regimens and/or radiation treatments. Anti-tumorantibodies, including, but not limited to anti-tumor antibody fragmentsand/or derivatives thereof, and monoclonal anti-tumor antibodies,fragments and/or derivatives thereof, are known in the art as isadministration of such antibody reagents in cancer therapy (e.g.,Rituxan® (rituximab); Herceptin® (trastuzumab)). Administration of oneor more additional therapeutic agents may occur before, after and/orconcurrent with administration of the IMPs.

Immunomodulatory therapy in accordance with the invention is also usefulfor individuals with infectious diseases, particularly infectiousdiseases which are resistant to humoral immune responses (e.g., diseasescaused by mycobacterial infections and intracellular pathogens).Immunomodulatory therapy may be used for the treatment of infectiousdiseases caused by cellular pathogens (e.g., bacteria or protozoans) orby subcellular pathogens (e.g., viruses). IMP therapy may beadministered to individuals suffering from mycobacterial diseases suchas tuberculosis (e.g., M. tuberculosis and/or M. bovis infections),leprosy (i.e., M. leprae infections), or M. marinum or M. ulceransinfections. IMP therapy is also useful for the treatment of viralinfections, including infections by influenza virus, respiratorysyncytial virus (RSV), hepatitis virus B, hepatitis virus C, herpesviruses, particularly herpes simplex viruses, and papilloma viruses.Diseases caused by intracellular parasites such as malaria (e.g.,infection by Plasmodium vivax, P. ovale, P. falciparum and/or P.malariae), leishmaniasis (e.g., infection by Leishmania donovani, L.tropica, L. mexicana, L. braziliensis, L. peruviana, L. infantum, L.chagasi, and/or L. aethiopica), and toxoplasmosis (i.e., infection byToxoplasmosis gondii) also benefit from IMP therapy. IMP therapy is alsouseful for treatment of parasitic diseases such as schistosomiasis(i.e., infection by blood flukes of the genus Schistosoma such as S.haematobium, S. mansoni, S. japonicum, and S. mekongi) and clonorchiasis(i.e., infection by Clonorchis sinensis). Administration of an IMP to anindividual suffering from an infectious disease results in anamelioration of symptoms of the infectious disease. In some embodiments,the infectious disease is not a viral disease.

The invention further provides methods of increasing or stimulating atleast one Th1-associated cytokine in an individual, including IL-2,IL-12, TNF-β, IFN-γ and IFN-α. In certain embodiments, the inventionprovides methods of increasing or stimulating IFN-γ in an individual,particularly in an individual in need of increased IFN-γ levels, byadministering an effective amount of an IMP to the individual such thatIFN-γ is increased. Individuals in need of increased IFN-γ are thosehaving disorders which generally respond to the administration of IFN-γ.Such disorders include a number of inflammatory disorders including, butnot limited to, ulcerative colitis. Such disorders also include a numberof fibrotic disorders, including, but not limited to, idiopathicpulmonary fibrosis (IPF), scleroderma, cutaneous radiation-inducedfibrosis, hepatic fibrosis including schistosomiasis-induced hepaticfibrosis, renal fibrosis as well as other conditions which may beimproved by administration of IFN-γ. Administration of IMP in accordancewith the invention results in an increase in IFN-γ levels, and resultsin amelioration of one or more symptoms, stabilization of one or moresymptoms, and/or prevention or slowing of progression (e.g., reductionor elimination of additional lesions or symptoms) of the disorder whichresponds to IFN-γ.

The methods of the invention may be practiced in combination with othertherapies which make up the standard of care for the disorder, such asadministration of anti-inflammatory agents such as systemiccorticosteroid therapy (e.g., cortisone) in IPF.

In certain embodiments, the invention provides methods of increasingtype I interferon, including IFN-α, IFN-β and IFN-ω, in an individual,particularly in an individual in need of increased type I interferonlevels, by administering an effective amount of an IMP to the individualsuch that type I interferon levels are increased. In certainembodiments, the invention provides methods of increasing IFN-α in anindividual, particularly in an individual in need of increased IFN-αlevels, by administering an effective amount of an IMP to the individualsuch that IFN-α levels are increased. Individuals in need of increasedIFN-α are those having disorders which generally respond to theadministration of IFN-α, including recombinant IFN-α, including, but notlimited to, viral infections and cancer. In some embodiments in whichincreased production of higher levels of IFN-α is desired, the IMPcontains at least one palindromic sequence of at least the followinglengths (in bases): 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, and,in some embodiments, the IMP contains at least one palindromic sequencewith a length longer than 30 bases.

Administration of IMP in accordance with the invention results in anincrease in IFN-α levels, and results in amelioration of one or moresymptoms, stabilization of one or more symptoms, and/or prevention orslowing of progression (e.g., reduction or elimination of additionallesions or symptoms) of the disorder which responds to IFN-α. Themethods of the invention may be practiced in combination with othertherapies which make up the standard of care for the disorder, such asadministration of anti-viral agents for viral infections.

Also provided are methods of reducing levels, particularly serum levels,of IgE in an individual having an IgE-related disorder by administeringan effective amount of an IMP to the individual. In such methods, theimmunomodulatory polynucleotide may be administered alone (e.g., withoutantigen) or administered with antigen, such as an allergen. Reduction inIgE results in an amelioration of one or more symptoms of theIgE-related disorder. Such symptoms include allergy symptoms such asrhinitis, conjunctivitis, in decreased sensitivity to allergens, areduction in the symptoms of allergy in an individual with allergies, ora reduction in severity of an allergic response. Accordingly, theinvention also provides methods of treating an allergic condition in anindividual. In some embodiments, methods of treating an allergiccondition include administering the immunomodulatory polynucleotide witha particular amount or dose of antigen. With any additional antigenadministration, the amount or dose of antigen administered can remainthe same, can decease or can increase (as in conventionaldesensitization therapy) over the course of treatment.

In some embodiments, the invention provides methods of stimulating CTLproduction in an individual, particularly in an individual in need ofincreased number and/or activity of CTLs, comprising administering aneffective amount of an IMP to the individual such that CTL production isincreased. Individuals in need of increased CTL production are thosehaving disorders which generally respond to CTL activity. Such disordersinclude, but not limited to, cancer and intracellular infections.Administration of IMP in accordance with the invention results in anincrease in CTL levels, and results in amelioration of one or moresymptoms, stabilization of one or more symptoms, and/or prevention orslowing of progression (e.g., reduction or elimination of additionallesions or symptoms) of the disorder which responds to CTL activity.

Methods of the invention include any embodiments described herein, suchas administering IMPs in the form of immunomodulatorypolynucleotide/microcarrier complex (with or without antigen, or with orwithout antigen over a course of administrations), or in proximateassociation with an antigen.

As will be apparent to one of skill in the art, the methods of theinvention may be practiced in combination with other therapies for theparticular indication for which the IMP is administered. For example,IMP therapy may be administered in conjunction with anti-malarial drugssuch as chloroquine for malaria patients, in conjunction withleishmanicidal drugs such as pentamidine and/or allopurinol forleishmaniasis patients, in conjunction with anti-mycobacterial drugssuch as isoniazid, rifampin and/or ethambutol in tuberculosis patients,or in conjunction with allergen desensitization therapy for atopic(allergy) patients.

As described herein, administration of IMPs may further compriseadministration of one or more additional immunotherapeutic agents (i.e.,an agent which acts via the immune system and/or is derived from theimmune system) including, but not limited to, cytokine, adjuvants andantibodies (including, but not limited to, antibody fragments and/orderivatives and monoclonal antibodies, fragments and/or derivativesthereof). Examples of therapeutic antibodies include those used in thecancer context (e.g., anti-tumor antibodies). Administration of suchadditional immunotherapeutic agents applies to all the methods describedherein.

An IMP may also be administered in conjunction with an adjuvant.Administration of an antigen with an IMP and an adjuvant leads to apotentiation of a immune response to the antigen and thus, can result inan enhanced immune response compared to that which results from acomposition comprising the IMP and antigen alone. Adjuvants are known inthe art and include, but are not limited to, oil-in-water emulsions,water-in oil emulsions, alum (aluminum salts), liposomes andmicroparticles, including but not limited to, polystyrene, starch,polyphosphazene and polylactide/polyglycosides. Other suitable adjuvantsalso include, but are not limited to, MF59, DETOX™ (Ribi), squalenemixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacteriumcell wall preparations, monophosphoryl lipid A, mycolic acidderivatives, nonionic block copolymer surfactants, Quil A, cholera toxinB subunit, polyphosphazene and derivatives, and immunostimulatingcomplexes (ISCOMs) such as those described by Takahashi et al. (1990)Nature 344:873-875, as well as, lipid-based adjuvants and othersdescribed herein. For veterinary use and for production of antibodies inanimals, mitogenic components of Freund's adjuvant (both complete andincomplete) can be used.

Administration and Assessment of the Immune Response

The IMP can be administered in combination with other pharmaceuticaland/or immunogenic and/or immunostimulatory agents, as described herein,and can be combined with a physiologically acceptable carrier thereof(and as such the invention includes these compositions). The IMP may beany of those described herein.

Accordingly, the IMP can be administered in conjunction with otherimmunotherapeutic agents including, but not limited to, cytokine,adjuvants and antibodies.

As with all immunogenic compositions, the immunologically effectiveamounts and method of administration of the particular IMP formulationcan vary based on the individual, what condition is to be treated andother factors evident to one skilled in the art. Factors to beconsidered include the antigenicity of antigen if administered, whetheror not the IMP will be administered with or covalently attached to anadjuvant, delivery molecule and/or antigen, route of administration andthe number of immunizing doses to be administered. Such factors areknown in the art and it is well within the skill of those in the art tomake such determinations without undue experimentation. A suitabledosage range is one that provides the desired modulation of immuneresponse (e.g., stimulation of IFN-α and/or IFN-γ). When an immuneresponse to an antigen is desired, a suitable dosage range is one thatprovides the desired modulation of immune response to the antigen.Generally, dosage is determined by the amount of IMP administered to thepatient, rather than the overall quantity of IMP-containing compositionadministered. Useful dosage ranges of the IMP, given in amounts of IMPdelivered, may be, for example, from about any of the following: 1 to500 μg/kg, 100 to 400 μg/kg, 200 to 300 μg/kg, 1 to 100 μg/kg, 100 to200 μg/kg, 300 to 400 μg/kg, 400 to 500 μg/kg. The absolute amount givento each patient depends on pharmacological properties such asbioavailability, clearance rate and route of administration.

The effective amount and method of administration of the particular IMPformulation can vary based on the individual patient, desired resultand/or type of disorder, the stage of the disease and other factorsevident to one skilled in the art. The route(s) of administration usefulin a particular application are apparent to one of skill in the art.Routes of administration include but are not limited to topical, dermal,transdermal, transmucosal, epidermal, parenteral, gastrointestinal, andnaso-pharyngeal and pulmonary, including transbronchial andtransalveolar. A suitable dosage range is one that provides sufficientIMP-containing composition to attain a tissue concentration of about1-10 μM as measured by blood levels. The absolute amount given to eachpatient depends on pharmacological properties such as bioavailability,clearance rate and route of administration.

As described herein, APCs and tissues with high concentration of APCsare preferred targets for the IMP. Thus, administration of IMP tomammalian skin and/or mucosa, where APCs are present in relatively highconcentration, is preferred.

The present invention provides IMP formulations suitable for topicalapplication including, but not limited to, physiologically acceptableimplants, ointments, creams, rinses and gels. Topical administration is,for instance, by a dressing or bandage having dispersed therein adelivery system, by direct administration of a delivery system intoincisions or open wounds, or by transdermal administration devicedirected at a site of interest. Creams, rinses, gels or ointments havingdispersed therein an IMP are suitable for use as topical ointments orwound filling agents.

Preferred routes of dermal administration are those which are leastinvasive. Preferred among these means are transdermal transmission,epidermal administration and subcutaneous injection. Of these means,epidermal administration is preferred for the greater concentrations ofAPCs expected to be in intradermal tissue.

Transdermal administration is accomplished by application of a cream,rinse, gel, etc. capable of allowing the IMP to penetrate the skin andenter the blood stream. Compositions suitable for transdermaladministration include, but are not limited to, pharmaceuticallyacceptable suspensions, oils, creams and ointments applied directly tothe skin or incorporated into a protective carrier such as a transdermaldevice (so-called “patch”). Examples of suitable creams, ointments etc.can be found, for instance, in the Physician's Desk Reference.

For transdermal transmission, iontophoresis is a suitable method.Iontophoretic transmission can be accomplished using commerciallyavailable patches which deliver their product continuously throughunbroken skin for periods of several days or more. Use of this methodallows for controlled transmission of pharmaceutical compositions inrelatively great concentrations, permits infusion of combination drugsand allows for contemporaneous use of an absorption promoter.

An exemplary patch product for use in this method is the LECTRO PATCHtrademarked product of General Medical Company of Los Angeles, Calif.This product electronically maintains reservoir electrodes at neutral pHand can be adapted to provide dosages of differing concentrations, todose continuously and/or periodically. Preparation and use of the patchshould be performed according to the manufacturer's printed instructionswhich accompany the LECTRO PATCH product; those instructions areincorporated herein by this reference. Other occlusive patch systems arealso suitable.

For transdermal transmission, low-frequency ultrasonic delivery is alsoa suitable method. Mitragotri et al. (1995) Science 269:850-853.Application of low-frequency ultrasonic frequencies (about 1 MHz) allowsthe general controlled delivery of therapeutic compositions, includingthose of high molecular weight.

Epidermal administration essentially involves mechanically or chemicallyirritating the outermost layer of the epidermis sufficiently to provokean immune response to the irritant. Specifically, the irritation shouldbe sufficient to attract APCs to the site of irritation.

An exemplary mechanical irritant means employs a multiplicity of verynarrow diameter, short tines which can be used to irritate the skin andattract APCs to the site of irritation, to take up IMP transferred fromthe end of the tines. For example, the MONO-VACC old tuberculin testmanufactured by Pasteur Merieux of Lyon, France contains a devicesuitable for introduction of IMP-containing compositions.

The device (which is distributed in the U.S. by Connaught Laboratories,Inc. of Swiftwater, Pa.) consists of a plastic container having asyringe plunger at one end and a tine disk at the other. The tine disksupports a multiplicity of narrow diameter tines of a length which willjust scratch the outermost layer of epidermal cells. Each of the tinesin the MONO-VACC kit is coated with old tuberculin; in the presentinvention, each needle is coated with a pharmaceutical composition ofIMP formulation. Use of the device is preferably according to themanufacturer's written instructions included with the device product.Similar devices which can also be used in this embodiment are thosewhich are currently used to perform allergy tests.

Another suitable approach to epidermal administration of IMP is by useof a chemical which irritates the outermost cells of the epidermis, thusprovoking a sufficient immune response to attract APCs to the area. Anexample is a keratinolytic agent, such as the salicylic acid used in thecommercially available topical depilatory creme sold by NoxemaCorporation under the trademark NAIR. This approach can also be used toachieve epithelial administration in the mucosa. The chemical irritantcan also be applied in conjunction with the mechanical irritant (as, forexample, would occur if the MONO-VACC type tine were also coated withthe chemical irritant). The IMP can be suspended in a carrier which alsocontains the chemical irritant or coadministered therewith.

Parenteral routes of administration include but are not limited toelectrical (iontophoresis) or direct injection such as direct injectioninto a central venous line, intravenous, intramuscular, intraperitoneal,intradermal, or subcutaneous injection. Formulations of IMP suitable forparenteral administration are generally formulated in USP water or waterfor injection and may further comprise pH buffers, salts bulking agents,preservatives, and other pharmaceutically acceptable excipients.Immunomodulatory polynucleotide for parenteral injection may beformulated in pharmaceutically acceptable sterile isotonic solutionssuch as saline and phosphate buffered saline for injection.

Gastrointestinal routes of administration include, but are not limitedto, ingestion and rectal. The invention includes formulations IMPsuitable for gastrointestinal administration including, but not limitedto, pharmaceutically acceptable powders, pills or liquids for ingestionand suppositories for rectal administration. As will be apparent to oneof skill in the art, pills or suppositories will further comprisepharmaceutically acceptable solids, such as starch, to provide bulk forthe composition.

Naso-pharyngeal and pulmonary administration include are accomplished byinhalation, and include delivery routes such as intranasal,transbronchial and transalveolar routes. The invention includesformulations of IMP suitable for administration by inhalation including,but not limited to, liquid suspensions for forming aerosols as well aspowder forms for dry powder inhalation delivery systems. Devicessuitable for administration by inhalation of IMP formulations include,but are not limited to, atomizers, vaporizers, nebulizers, and drypowder inhalation delivery devices.

As is well known in the art, solutions or suspensions used for theroutes of administration described herein can include any one or more ofthe following components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

As is well known in the art, pharmaceutical compositions suitable forinjectable use include sterile aqueous solutions (where water soluble)or dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. For intravenousadministration, suitable carriers include physiological saline,bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It should be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. It may be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol,sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

As is well known in the art, sterile injectable solutions can beprepared by incorporating the active compound(s) in the required amountin an appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

The choice of delivery routes can be used to modulate the immuneresponse elicited. For example, IgG titers and CTL activities wereidentical when an influenza virus vector was administered viaintramuscular or epidermal (gene gun) routes; however, the muscularinoculation yielded primarily IgG2a, while the epidermal route yieldedmostly IgG1. Pertmer et al. (1996) J. Virol. 70:6119-6125. Thus, oneskilled in the art can take advantage of slight differences inimmunogenicity elicited by different routes of administering theimmunomodulatory polynucleotides of the present invention.

The above-mentioned compositions and methods of administration are meantto describe but not limit the methods of administering the formulationsof IMPs of the invention. The methods of producing the variouscompositions and devices are within the ability of one skilled in theart and are not described in detail here.

Analysis (both qualitative and quantitative) of the immune response toIMP can be by any method known in the art, including, but not limitedto, measuring antigen-specific antibody production (including measuringspecific antibody subclasses), activation of specific populations oflymphocytes such as CD4+ T cells, B cells, NK cells or CTLs, maturationof dendritic cells (including plasmacytoid dendritic cells), productionof cytokines and chemokines such as IFN-γ, IFN-α, IFN-ω, TNF-α, IL-2,IL-4, IL-5, IL-6, IL-10, IL-12, IP-10, MCP-1, MCP-2, MCP-3, MIG orMIP-3β and/or release of histamine. Methods for measuring specificantibody responses include enzyme-linked immunosorbent assay (ELISA) andare well known in the art. Measurement of numbers of specific types oflymphocytes such as CD4+ T cells can be achieved, for example, withfluorescence-activated cell sorting (FACS). Measurement of activation ofparticular populations of cells can be achieved by determiningexpression of markers, for example, cell surface markers, specific foractivation of the particular cell type. Cell marker expression can bemeasured, for example, by measuring RNA expression or measuring cellsurface expression of the particular marker by, for example, FACSanalysis. Cytotoxicity and CTL assays can be performed for instance asdescribed in Raz et al. (1994) Proc. Natl. Acad. Sci. USA 91:9519-9523and Cho et al. (2000). Cytokine concentrations can be measured, forexample, by ELISA. Measuring maturation of dendritic cells can beperformed for instance as described in Hartmann et al. (1999) Proc.Natl. Acad. Sci. USA 96:9305-9310. These and other assays to evaluatethe immune response to an immunogen are well known in the art. See, forexample, Selected Methods in Cellular Immunology (1980) Mishell andShiigi, eds., W.H. Freeman and Co.

Analysis (both qualitative and quantitative) of the immune response toIMP can also be by measuring the level of cytokines, chemokines and/orother molecules that are induced by cytokines, such as IFN-γ and/orIFN-α, whose production is stimulated by IMP. Accordingly, the IMPs ofthe invention may also stimulate expression of IFN-γ and/or IFN-αinducible cytokines, chemokines and inflammatory proteins including, butnot limited to, IP-10 (interferon induced protein 10 kDa), monokineinduced by IFN-γ, and monocyte chemotactic protein 1 (MCP-1). The immuneresponse to IMP can also be analyzed by measuring the level ofcytokines, chemokines and/or other molecules that are known to haveantiviral activities, including 2,5-oligoadenylate synthetase (2,5-OAS),interferon-stimulating gene-54K (ISG-54K), MxA, MxB andguanylate-binding protein-1 (GBP-1). Thus, antiviral molecules andmolecules induced by IFN-γ and/or IFN-α can be used as markers of IMPactivity. Measurement of such interferon-induced molecule productionand/or gene expression can be by any method known in the art, including,but not limited to, by ELISA and quantitative PCR to measure RNAproduction.

Preferably, a Th1-type response is stimulated, i.e., elicited and/orenhanced. With reference to the invention, stimulating a Th1-type immuneresponse can be determined in vitro or ex vivo by measuring cytokineproduction from cells treated with IMP as compared to those treatedwithout IMP. Methods to determine the cytokine production of cellsinclude those methods described herein and any known in the art. Thetype of cytokines produced in response to IMP treatment indicate aTh1-type or a Th2-type biased immune response by the cells. As usedherein, the term “Th1-type biased” cytokine production refers to themeasurable increased production of cytokines associated with a Th1-typeimmune response in the presence of a stimulator as compared toproduction of such cytokines in the absence of stimulation. Examples ofsuch Th1-type biased cytokines include, but are not limited to, IL-2,IL-12, IFN-γ and IFN-α. In contrast, “Th2-type biased cytokines” refersto those associated with a Th2-type immune response, and include, butare not limited to, IL-4, IL-5, and IL-13. Cells useful for thedetermination of IMP activity include cells of the immune system,primary cells isolated from a host and/or cell lines, preferably APCsand lymphocytes, even more preferably macrophages and T cells.

Stimulating a Th1-type immune response can also be measured in a hosttreated with an IMP can be determined by any method known in the artincluding, but not limited to: (1) a reduction in levels of IL-4 or IL-5measured before and after antigen-challenge; or detection of lower (oreven absent) levels of IL-4 or IL-5 in an IMP treated host, optionallyas compared to an antigen-primed, or primed and challenged, controltreated without IMP; (2) an increase in levels of IL-12, IL-18 and/orIFN (α, β or γ) before and after antigen challenge; or detection ofhigher levels of IL-12, IL-18 and/or IFN (α, β or γ) in an IMP treatedhost as compared to an antigen-primed or, primed and challenged, controltreated without IMP; (3) “Th1-type biased” antibody production in an IMPtreated host as compared to a control treated without IMP; and/or (4) areduction in levels of antigen-specific IgE as measured before and afterantigen challenge; or detection of lower (or even absent) levels ofantigen-specific IgE in an IMP treated host as compared to anantigen-primed, or primed and challenged, control treated without IMP. Avariety of these determinations can be made by measuring cytokines madeby APCs and/or lymphocytes, preferably macrophages and/or T cells, invitro or ex vivo using methods described herein or any known in the art.Some of these determinations can be made by measuring the class and/orsubclass of antigen-specific antibodies using methods described hereinor any known in the art.

The class and/or subclass of antigen-specific antibodies produced inresponse to IMP treatment indicate a Th1-type or a Th2-type biasedimmune response by the cells. As used herein, the term “Th1-type biased”antibody production refers to the measurable increased production ofantibodies associated with a Th1-type immune response (i.e.,Th1-associated antibodies). One or more Th1 associated antibodies may bemeasured. Examples of such Th1-type biased antibodies include, but arenot limited to, human IgG1 and/or IgG3 (see, e.g., Widhe et al. (1998)Scand. J. Immunol. 47:575-581 and de Martino et al. (1999) Ann. AllergyAsthma Immunol. 83:160-164) and murine IgG2a. In contrast, “Th2-typebiased antibodies” refers to those associated with a Th2-type immuneresponse, and include, but are not limited to, human IgG2, IgG4 and/orIgE (see, e.g., Widhe et al. (1998) and de Martino et al. (1999)) andmurine IgG1 and/or IgE.

The Th1-type biased cytokine induction which occurs as a result ofadministration of IMP produces enhanced cellular immune responses, suchas those performed by NK cells, cytotoxic killer cells, Th1 helper andmemory cells. These responses are particularly beneficial for use inprotective or therapeutic vaccination against viruses, fungi, protozoanparasites, bacteria, allergic diseases and asthma, as well as tumors.

In some embodiments, a Th2 response is suppressed (reduced). Suppressionof a Th2 response may be determined by, for example, reduction in levelsof Th2-associated cytokines, such as IL-4 and IL-5, reduction in thelevels of Th2-associated antibodies, as well as IgE reduction andreduction in histamine release in response to allergen.

Kits of the Invention

The invention provides kits. In certain embodiments, the kits of theinvention generally comprise one or more containers comprising any IMPas described herein. The kits may further comprise a suitable set ofinstructions, generally written instructions, relating to the use of theIMP for any of the methods described herein (e.g., immunomodulation,ameliorating one or more symptoms of an infectious disease, increasingIFN-γ levels, increasing IFN-α levels, or ameliorating an IgE-relateddisorder).

The kits may comprise IMP packaged in any convenient, appropriatepackaging. For example, if the IMP is a dry formulation (e.g., freezedried or a dry powder), a vial with a resilient stopper is normallyused, so that the IMP may be easily resuspended by injecting fluidthrough the resilient stopper. Ampoules with non-resilient, removableclosures (e.g., sealed glass) or resilient stoppers are mostconveniently used for liquid formulations of IMP. Also contemplated arepackages for use in combination with a specific device, such as aninhaler, nasal administration device (e.g., an atomizer) or an infusiondevice such as a minipump.

The instructions relating to the use of IMP generally includeinformation as to dosage, dosing schedule, and route of administrationfor the intended method of use. The containers of IMP may be unit doses,bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

In some embodiments, the kits further comprise an antigen (or one ormore antigens), which may or may not be packaged in the same container(formulation) as the IMP(s). Antigen have been described herein.

In certain embodiments, the kits of the invention comprise an IMP in theform of an immunomodulatory polynucleotide/microcarrier complex (IMP/MC)and may further comprise a set of instructions, generally writteninstructions, relating to the use of the IMP/MC complex for any of themethods described herein (e.g., immunomodulation, ameliorating one ormore symptoms of an infectious disease, increasing IFN-γ levels,increasing IFN-α levels, or ameliorating an IgE-related disorder).

In some embodiments, kits of the invention comprise materials forproduction of IMP/MC complex generally include separate containers ofIMP and MC, although in certain embodiments materials for producing theMC are supplied rather than preformed MC. The IMP and MC are preferablysupplied in a form which allows formation of IMP/MC complex upon mixingof the supplied IMP and MC. This configuration is preferred when theIMP/MC complex is linked by non-covalent bonding. This configuration isalso preferred when the IMP and MC are to be crosslinked via aheterobifunctional crosslinker; either IMP or the MC is supplied in an“activated” form (e.g., linked to the heterobifunctional crosslinkersuch that a moiety reactive with the IMP is available).

Kits for IMP/MC complexes comprising a liquid phase MC preferablycomprise one or more containers including materials for producing liquidphase MC. For example, an IMP/MC kit for oil-in-water emulsion MC maycomprise one or more containers containing an oil phase and an aqueousphase. The contents of the container are emulsified to produce the MC,which may be then mixed with the IMP, preferably an IMP which has beenmodified to incorporate a hydrophobic moiety. Such materials include oiland water, for production of oil-in-water emulsions, or containers oflyophilized liposome components (e.g., a mixture of phospholipid,cholesterol and a surfactant) plus one or more containers of an aqueousphase (e.g., a pharmaceutically-acceptable aqueous buffer). In certainembodiments, the kits of the invention comprise an IMP in the form of acationic condensing agent—IMP—stabilizing agent (CIS) composition in oneor more containers comprising any immunomodulatory CIS particulatecomposition as described herein. Alternately, the kits may comprise oneor more containers of the components of the CIS compositions of theinvention. Configurations of this embodiment include kits with acontainer of IMP/stabilizing agent mixture and a container of cationiccondensing agent and kits with a container of IMP, a container ofstabilizing agent, and a container of cationic condensing agent. Thekits may further comprise a suitable set of instructions, generallywritten instructions, relating to the use of the CIS particulatecomposition for any of the methods described herein (e.g.,immunomodulation, ameliorating one or more symptoms of an infectiousdisease, increasing IFN-γ levels, increasing IFN-α levels, orameliorating an IgE-related disorder). The kit embodiments that comprisecontainers of the components of the CIS compositions will generallyinclude instructions for production of the CIS compositions inaccordance with the methods disclosed herein. In addition to the CIScomposition and/or components of the CIS composition of the invention,kit embodiments may also enclose instructions for production of the CIScompositions in accordance with the methods disclosed herein andinstructions for use of the immunomodulatory CIS compositions for any ofthe methods described herein.

The following Examples are provided to illustrate, but not limit, theinvention.

EXAMPLES Example 1 Immunomodulation of Human Cells by ImmunomodulatoryPolynucleotides

Immunomodulatory polynucleotides (IMPs) or control samples, includingpolynucleotides without an immunomodulatory sequence(5′-TGACTGTGAACCTTAGAGATGAquew-3′ (SEQ ID NO: 2)), SAC and media alone,were tested for immunomodulatory activity on human peripheral bloodmononuclear cells (PBMCs). Also tested was the standard immunomodulatorypolynucleotide 5′-TGACTGTGAACGTTCGAGATGA. (SEQ ID NO:1). Unless notedotherwise, the polynucleotides tested were fully modifiedphosphorothioate oligodeoxynucleotides.

Peripheral blood was collected from volunteers by venipuncture usingheparinized syringes. Blood was layered onto a FICOLL® (AmershamPharmacia Biotech) cushion and centrifuged. PBMCs, located at theFICOLL® interface, were collected, then washed twice with cold phosphatebuffered saline (PBS). The cells were resuspended and cultured in 48 or96 well plates at 2×10⁶ cells/mL in RPMI 1640 with 10% heat-inactivatedhuman AB serum plus 50 units/mL penicillin, 50 μg/mL streptomycin, 300μg/mL glutamine, 1 mM sodium pyruvate, and 1×MEM non-essential aminoacids (NEAA).

The cells were cultured in the presence of test samples (IMPs orcontrols) at doses ranging from 0.2 to 20 μg/ml for 24 hours, thencell-free medium was collected from each well and assayed for IFN-γand/or IFN-α concentration. IFN-γ and IFN-α were assayed usingCYTOSCREEN™ ELISA kits from BioSource International, Inc., according tothe manufacturer's instructions. Generally, the test samples were testedwith PBMCs from 4 human donors.

IMPs stimulated IFN-γ and/or IFN-α secretion by human PBMCs. In thehuman PBMC assay, background levels of IFN-γ can vary, evensignificantly, with the donor. Other cytokines such as IFN-α, however,demonstrate a generally stable pattern of activation and routinelyexhibit low background levels under unstimulated conditions. Examples ofresults from such assays with PBMCs are summarized in Tables 2-7.

In a dose titration assay, PBMCs from 4 donors were stimulated with 0.2to 20 μg/ml of SEQ ID NO:27 as described above. The amount of IFN-α andIFN-γ produced was assessed as described above and the results from the4 donors were averaged and the mean results are presented in Table 2.

TABLE 2 IMP titration - IFN (pg/ml) SEQ ID NO: 27 (μg/ml) IFN-γ IFN-α 20412 749 8 583 4036 3.2 203 4073 1.3 39 887 0.5 15 108 0.2 11 50

As can be seen from the results presented in Table 2, the capability toinduce IFN-α production increased as the IMP dose decreased and becameoptimal at approximately 3-8 μg/ml, after which the activity decreasedwith dose. Additional assays confirmed this result.

PBMCs from four donors were stimulated with 20 μg/ml of IMPs or controlsand the stimulation of IFN-α and IFN-γ production was assessed asdescribed above. Among the polynucleotides tested were:

5′-TCGTCGAACGTTCGTTAACGTTCG; (SEQ ID NO: 5) 5′-TCGTCGAACGTTCGTT;(SEQ ID NO: 12) 5′-TCGTCGGAACGTTCGAGATG; (SEQ ID NO: 14)5′-TCGTCGTGAACGTTCGAGATGA; (SEQ ID NO: 13) 5′-TCGTCGAACGTTCCTTAACGTTCC;(SEQ ID NO: 6) 5′-TCGTCGTAACGTTCGAGATG; (SEQ ID NO: 15)5′-TCGTCGAACGTTTTAACGTT; (SEQ ID NO: 31) 5′-TCGTTCAACGTTCGTTAACGTTCG;(SEQ ID NO: 9) 5′-TCGTCGGACGTTCGAGATG; (SEQ ID NO: 16)5′-TCGTCGTACGTTCGAGATG; (SEQ ID NO: 17) 5′-TCGTCGTTCGTTCGAGATG;(SEQ ID NO: 18) 5′-TCGTCGAACCTTCGTTAACCTTCG; (SEQ ID NO: 11)5′-TGATCGTCGAACGTTCGAGATG; (SEQ ID NO: 24) 5′-TGATCGAACGTTCGTTAACGTTCG;(SEQ ID NO: 8) 5′-TGATTCAACGTTCGTTAACGTTCG; (SEQ ID NO: 10)5′-TCAACGTTCGTTAACGTTCGTT. (SEQ ID NO: 4)

The results of cytokine production from the PBMCs from each donor wasaveraged and the mean results are presented in Table 3.

TABLE 3 Human PBMC Assays - IFN (pg/ml) test or control (SEQ ID NO.)IFN-γ IFN-α 2 (non-IMP) 11 50 1 (IMP std) 205 141 27 335 842  5 297 51735 308 686 12 153 157 14 340 576 13 297 142  7 510 594  6 554 103 15 204194 31 169 178  9 310 57 16 274 421 17 387 208 18 78 50 11 36 50 24 462708  8 650 704 10 111 66  4 126 50 media 11 50

As demonstrated in Table 3, IMPs that stimulated production of moreIFN-α than an IMP standard, SEQ ID NO: 1, include at least one TCGsequence at or near the 5′ end of the polynucleotide (a 5′-TCG sequence)and a palindromic sequence of at least 8 bases in length either adjacentto or within 3 bases of the 5′-TCG sequence. In general, stimulation ofIFN-γ production mirrored stimulation of IFN-α production, although therange of variation in the IFN-γ stimulation was less than for IFN-α. Inthe polynucleotides in which the palindromic sequence and the 5′-TCGwere separated, it was generally preferable for the production of IFN-αthat the separation be by or overlapping with a second TCG trinucleotide(see, for example, SEQ ID NO:14). IMPs containing a 5′-TCG but nopalindromic sequence as described above induced very low levels of IFN-γand did not induce IFN-α production (see, for example, SEQ ID NOs: 18and 11). IMPs containing 6-8 base palindromes but no 5′-TCGtrinucleotide induced IFN-γ but only low levels of IFN-α (see, forexample, SEQ ID NO: 1 and 4). Notably, IMPs containing a TCG up to threebases removed from the 5′ end of the polynucleotide and containing apalindromic sequence of at least 10 bases in length induced aparticularly high level of IFN-α compared to an IMP standard without a5′-TCG, SEQ ID NO: 1 (see, for example, SEQ ID NO: 24 and 8).

An assay was performed to test IMP dose dependence on stimulation ofIFN-α production. IMPs tested in this assay varied in the position ofthe palindromic sequence in the polynucleotide and/or the position ofthe at least one TCG sequence at the 5′ end. Among the polynucleotidestested were some with CG dinucleotides and 5′-TCG sequences but withoutpalindromic sequences 8 bases or greater in length (for example, SEQ IDNO:11; SEQ ID NO:18; 5′-TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO:3)). Alsotested were polynucleotides with CG dinucleotides and palindromicsequences 8 bases or greater in length but no 5′-TCG trinucleotides (forexample, SEQ ID NO:1; SEQ ID NO:4; 5′-ATCATCTCGAACGTTCGACGA (SEQ IDNO:29); 5′-AACGTTCGAACGTTCGAACGTTT (SEQ ID NO:67);5′-TCAACGTTCGAACGTTCGAACGTT (SEQ ID NO:68); 5′-GACGATCGTCGACGATCGTC (SEQID NO:85)). PBMCs from four donors were stimulated with either 0.8, 4.0or 20 μg/ml of IMPs or controls. The stimulation of IFN-α production wasassessed as described above and the results averaged from the 4 donorsare reported in Table 4.

TABLE 4 Human PBMC Assays - IFN-α (pg/ml) test or control (SEQ ID NO.)20 μg/ml 4.0 μg/ml 0.8 μg/ml 2 (non-IMP) 52 52 52 1 (IMP std) 52 108 5227 8626 7908 715 52 2425 4249 1085 39 2388 9325 3590 38 1874 7744 463557 1991 4262 9780 58 915 1654 5965 59 616 3221 1147 24 1848 2233 71  81023 544 52 29 1000 3325 95 35 3507 8734 63 60 1978 517 52 61 7256 13767599 62 11157 16722 2254 63 17077 12510 360 64 569 2896 80 65 2007 115855 66 3926 718 64 67 246 2399 52 68 520 1558 1254 85 52 411 52  4 158124 52 18 473 618 52 11 52 261 756  3 138 289 53 medium 52

The results presented in Table 4 support the importance of a palindromicsequence at least 8 bases in length and at least one TCG sequence at ornear the 5′ end of the polynucleotide for stimulation of IFN-α fromhuman PBMCs.

Another assay was performed to test IMP dose dependence on stimulationof IFN-⋆ production. IMPs tested in this assay varied in the presence ofCG dinucleotides and 5′-TCG sequences in the polynucleotide. Among thepolynucleotides tested were some with palindromic sequences but withoutCG dinucleotides (for example, SEQ ID NO:2; 5′-TGCTTGCAAGCTTGCAAGCA (SEQID NO: 90), 5′-TCAGTCAGTCAGCTGACTGACTGA (SEQ ID NO:96) and/or without a5′-TCG sequence (for example, SEQ ID NOs:1, 90, 96;5′-ACCGATAACGTTGCCGGTGACGGCACCACG (SEQ ID NO:92), 5′-AACAACAACGTTGTTGTT(SEQ ID NO:95), 5′-ACCGATAACGTTGCCGGTGACGGCACCACG (SEQ ID NO:25),5′-AACAACAACGTTGTTGTT (SEQ ID NO:94)). Also tested in this assay was thepolynucleotide 5′-TCGTTGCAAGCTTGCAACGA (SEQ ID NO:91). Some of the IMPsvaried in phosphate backbone composition. PBMCs from three donors werestimulated with either 0.8, 4.0 or 20 μg/ml of IMPs or controls. Thestimulation of IFN-α production was assessed using PBMCs from 3 donorsas described above and the averaged results for the 3 donors arereported in Table 5.

TABLE 5 Human PBMC Assays - IFN-α (pg/ml) test or control (SEQ ID NO.)20 μg/ml 4.0 μg/ml 0.8 μg/ml media 43 — — 2 (non-IMP) 43 43 43 1 (IMPstd) 43 371 43 27 823 4958 1893 53 1968 13779 13550 54 142 5090 2832 971244 12097 5173 42 1790 7923 4249 90 43 43 50 96 58 613 43 91 1177 1539870 25 43 43 43 92 43 903 43 94 235 56 43 95 216 84 43 26 25420 199034136 30 1125 7543 5955 32 1483 5088 2933 33 6031 24061 14111 34 1501217241 6979 93 1355 6193 1762

As can be seen from the results presented in Table 5, inversion of theCG dinucleotides in the highly active sequence SEQ ID NO:42 to GCdinucleotides abolishes the ability of SEQ ID NO:90 to induce IFN-α.Similarly SEQ ID NO:96, a palindromic polynucleotide without CGdinucleotides is also inactive.

As can be seen in Table 5, two representative phosphodiesterpolynucleotides, SEQ ID NO:25 and SEQ ID NO:94, and their fully modifiedphosphorothioate versions, SEQ ID NO:92 and SEQ ID NO:95, respectively,were not active in inducing IFN-α from human PBMCs. Although SEQ IDNOs:25 and 92 contain several CG dinucleotides, including the motifAACGTT, they do not include TCG or a palindromic sequence of at least 8bases. SEQ ID NOs:94 and 95 are 18 base palindromes and contain one CGdinucleotide, but no TCG trinucleotide. Thus, these polynucleotides donot fit the motifs described herein.

SEQ ID NOs:26, 30, 32, and 33, containing all phosphorothioate linkages(SEQ ID NOs:30 and 32) or chimeric phosphorothioate/phosphodiesterlinkages (SEQ ID NOs:26 and 33), induced high amounts of IFN-α fromhuman PBMCs. Both SEQ ID NOs:34 (which contains chimericphosphorothioate/phosphodiester linkages) and 93 (all phosphorothioatelinkages) induced IFN-α from human PBMCs.

In an assay to test the effect of the length of a palindromic sequenceon stimulation of IFN-α, PBMCs from four donors were stimulated witheither 2 μg/ml or 20 μg/ml of IMPs or controls, the stimulation of IFN-αproduction was assessed as described above and the averaged results arereported in Table 6. Among the polynucleotides tested were5′-TTCGAACGTTCGTTAACGTTCG (SEQ ID NO:20) and 5′-TCGTCGAACGTTCGAACGTTCG(SEQ ID NO:19).

TABLE 6 Human PBMC Assays - IFN-α (pg/ml) test or control (SEQ ID NO.)20 μg/ml 2 μg/ml 2 (non-IMP) 26 26 1 (IMP std) 93 34  5 2146 4018 202350 312 19 9844 15989 38 1935 15217 39 3729 14127 40 4584 12550 43 417410362 27 2008 10062 41 543 12916 42 3935 14752 media 26 26

The results presented in Table 6 support the importance of a palindromicsequence at least 8 bases in length and at least one TCG sequence at ornear the 5′ end of the polynucleotide for stimulation of IFN-α fromhuman PBMCs.

In an assay to test IFN-α stimulatory activity of IMPs with a variety of12 base palindromes, PBMCs from four donors were stimulated with either0.8, 4 or 20 μg/ml of IMPs or controls, the stimulation of IFN-αproduction was assessed as described above and the averaged results arereported in Table 7.

TABLE 7 Human PBMC Assays - IFN-α (pg/ml) test or control (SEQ ID NO.)20 μg/ml 4 μg/ml 0.8 μg/ml 2 (non-IMP) 169 133 133 1 (IMP std) 190 238143 27 3010 6473 2775 44 4951 10420 5468 45 3821 7221 2864 46 1403 52965169 47 2798 6731 3992 48 3082 9190 4113 51 2701 5699 1727 69 1886 82995195 70 7893 8429 5553 71 10647 10525 6173 72 9652 9101 5095 73 104199376 4896 74 9883 9085 5635 75 10269 8153 3888 76 10551 9773 5062 495424 7762 2788 50 6112 8517 3239 42 7634 8208 5472 43 6777 6768 4472 773694 4725 768 78 2542 4257 4311 79 1201 5725 5757 39 7454 9965 6622 802938 4137 1412 81 5914 4918 865 82 3451 4249 4170 84 3454 5363 2255 8610742 11881 6332 87 5110 5950 4139 114  4779 5491 2907 media 204 204 204

The results presented in Table 7 indicate that any of the IMPs testedwith 12 base palindromes were active in stimulating IFN-α from humanPBMCs. These IMPs contain a 12 base palindrome with the sequenceTCGX₁X₂CGX₂′X₁′CGA (SEQ ID NO:198) in which there are no nucleotidelimitations for X₁ and X₂, despite the formation of runs of CGCG, CCGGand GCGC, which have previously been described as immunoinhibitorysequences or immune neutralizing sequences (Krieg et al. (1998) Proc.Natl. Acad. Sci. USA 95:12631-12636). For example, SEQ ID NOs:49 and 50are active in stimulating IFN-α and contain the sequence CGCG. SEQ IDNO:49 exemplifies an immunomodulatory polynucleotide containing SEQ IDNO:161 described above. SEQ ID NO:50 exemplifies an immunomodulatorypolynucleotide containing SEQ ID NO:162 described above.

IMPs with longer palindromes induced higher levels of IFN-α from humanPBMCs, particularly at lower IMP doses. As can be seen in assay resultsshown in FIG. 1, the amount of IFN-α produced from the cells in responseto SEQ ID NO:172 was significantly higher than SEQ ID NO:113, SEQ IDNO:27 and SEQ ID NO:1 at the 0.4 μg/ml dose of IMP. Also, the amount ofIFN-α produced in response to SEQ ID NO:172 was significantly higherthan SEQ ID NO:27 and SEQ ID NO:1 at the 0.8 μg/ml dose of IMP(p<0.001). The palindrome length in the IMPs is: 28 bases in SEQ IDNO:172, 22 bases in SEQ ID NO:113, 12 bases in SEQ ID NO:27, and 8 basesin SEQ ID NO:1

In another assay, overall IMP length and IMP palindrome length werecompared in the induction of IFN-α production from human PBMCs. Amongthe polynucleotides tested were:

SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 27,5′-TCGTCGAACGTTCGAGATG; (SEQ ID NO: 166) 5′-TCGTCGAACGTTCGAGAT;(SEQ ID NO: 99) 5′-TCGTCGAACGTTCGAG; (SEQ ID NO: 100)5′-TCGTCGAACGTTCGA; (SEQ ID NO: 101) 5′-TCGAACGTTCGAG; (SEQ ID NO: 102)5′-TCGAACGTTCGA; (SEQ ID NO: 103) 5′-TCGAACGTTCG; (SEQ ID NO: 104)5′-TCGACGTCGA; (SEQ ID NO: 105) 5′-TCGTCGAACGTTCG; (SEQ ID NO: 167)5′-TCGTCGAACGTT; (SEQ ID NO: 199) 5′-TCGTTCGAACGTTCGAA; (SEQ ID NO: 54)5′-TTCGAACGTTCGAA. (SEQ ID NO: 98)PBMCs from four donors were stimulated with either 0.8, 4.0 or 20 μg/mlof IMPs or controls and the resultant production of IFN-α was assessedas described above. The averaged result for the 4 donors at each IMPconcentration are reported in Table 8.

TABLE 8 Human PBMC Assays - IFN-α (pg/ml) IMP Total test (SEQ ID IFN-αlength Palindrome NO.) or control 20 μg/ml 4 μg/ml 0.8 μg/ml (bases)(bases) 1 (IMP std) 128 412 52 22 8 2 (non-IMP) 52 52 52 22 —  27 11815697 1264 21 12 166 1527 4827 2095 19 12  99 204 4254 2093 18 12 100 4513835 2115 16 12 101 601 3065 547 15 12 102 1016 3529 533 13 12 103 4841091 83 12 12 104 321 52 52 11 10 105 52 52 52 10 10  12 224 1692 63 1610 167 319 556 69 14 10 199 52 52 52 12 6  54 99 3143 1133 17 14  981027 2321 744 14 14 media 82 82 82

The results presented in Table 8 indicate that, for the polynucleotidestested, the minimum total length of the polynucleotide to stimulateIFN-α production in human PBMCs is about 12 bases with a palindrome ofabout 10 bases in length. Accordingly, in some embodiments in whichproduction of higher levels of IFN-α is desired, the IMP contains atleast one palindromic sequence of at least the following lengths (inbases): 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, and, in someembodiments, the IMP contains at least one palindromic sequence with alength longer than 30 bases.

In another assay, PBMCs from three donors were stimulated with either0.8, 4.0 or 20 μg/ml of IMPs or controls. The stimulation of IFN-α,IFN-β and IFN-ω production was assessed as described above. IFN-ω wasassayed using an ELISA kit from PBL Biomedical Laboratories and thelower and upper limit of detection of IFN-ω was 48 pg/ml and 6000 pg/ml,respectively. IFN-β was assayed using an ELISA kit from BioSource andthe lower and upper limit of detection was 12 IU/ml and 3046 IU/ml,respectively. The averaged result for the 3 donors at each IMPconcentration are reported in Table 9.

TABLE 9 Human PBMC Assays - IFN-α or IFN-ω (pg/ml) IFN-α IFN-ω test (SEQID 20 4.0 0.8 20 4.0 0.8 NO.) or control μg/ml μg/ml μg/ml μg/ml μg/mlμg/ml media 16 — — 48 — — 2 (non-IMP) 14 16 14 48 48 48 1 (IMP std) 49198 19 48 48 48 27 700 7394 2146 76 629 163 39 2716 6180 5922 284 741604 38 nd nd nd 228 632 650 nd = not determined

As can been seen in Table 9, IMPs of the present invention stimulateproduction of IFN-ω from human PBMCs as well as production of IFN-α. Inthe assay described above, IFN-β was not detected.

In another assay, duplex forms of polynucleotides were compared tonon-duplex forms in the induction of IFN-α production from human PBMCs.Among the polynucleotides tested were: SEQ ID NO:1, SEQ ID NO:90, SEQ IDNO:27, and 5′-TCGTCGAACGTTCGAGATGAT/5′-ATCATCTCGAACGTTCGACGA (a duplexof SEQ ID NO:27 and SEQ ID NO:29). PBMCs from three donors werestimulated with either 0.4, 0.8, 4.0 or 20 μg/ml of IMPs or controls andthe resultant production of IFN-α was assessed as described above. Theduplexes were compared to the single sequences using the same total doseof polynucleotide (e.g., 4 μg/ml of SEQ ID NO:27 was compared to 4 μg/mlof double strand which contained 2 μg/ml SEQ ID NO:27 and 2 μg/ml SEQ IDNO:29). The averaged result for the 3 donors at each IMP concentrationare reported in Table 10.

TABLE 10 Human PBMC Assays - IFN-α (pg/ml) test (SEQ ID NO.) IFN-α orcontrol 20 μg/ml 4 μg/ml 0.8 μg/ml 0.4 μg/ml 27 592 3719 254 57 182(27/29 dpx) 386 2612 4725 1027  1 124 312 52 52 90 52 nd nd nd medium 5252 52 52 nd = not determined

As can been seen in Table 10, SEQ ID NO:182, the duplex form of SEQ IDNO:27 is more active than SEQ ID NO:27 in stimulating IFN-α productionat lower IMP doses. At higher doses (4 and 20 μg/ml), SEQ ID NO:27 wassomewhat more stimulatory.

In another assay, a polynucleotide containing modified bases andpolynucleotides without modified bases were compared in the induction ofIFN-α production from human PBMCs. Among the polynucleotides testedwere: SEQ ID NO:1, SEQ ID NO:2,5′-TCGTCGAACGTTCGAGATGAT (SEQ ID NO:27),and 5′-TCXTCXAACXTTCXAGATGAT (X=7-deaza-dG, SEQ ID NO:193). SEQ ID NO:27and SEQ ID NO:193 have the same nucleotide sequence except for thedeaza-dG substitutions for four dGs in SEQ ID NO:27. PBMCs from fourdonors were stimulated with either 0.8, 4.0 or 20 μg/ml of IMPs orcontrols and the resultant production of IFN-α was assessed as describedabove. The averaged result for the 4 donors at each IMP concentrationare reported in Table 11.

TABLE 11 Human PBMC Assays - IFN-α (pg/ml) test (SEQ ID IFN-α NO.) orcontrol 20 μg/ml 4 μg/ml 0.8 μg/ml 1 (IMP std) 129 118 80 2 (non-IMP)102 102 102  27 10248 13871 3798 193 10754 12262 193 medium 102

As can been seen in Table 11, SEQ ID NO:193 has IFN-α stimulatoryactivity comparable to SEQ ID NO:27 except at the 0.8 μg/ml dose.

Single and double strand forms of polynucleotides containing modifiedbases were assayed for activity in the induction of IFN-α productionfrom human PBMCs. Among the polynucleotides tested were: single anddouble strand SEQ ID NO:1, single strand SEQ ID NO:2, single strand SEQID NO:29, single strand and double strand SEQ ID NO:27, single anddouble strand SEQ ID NO:187, single and double strand SEQ ID NO:188,single and double strand SEQ ID NO:189, single and double strand SEQ IDNO:190, single strand SEQ ID NO:194, and single strand SEQ ID NO:197.SEQ ID NOs: 187, 188, 189, 190, 194 and 197 have the same nucleotidesequence as SEQ ID NO:27 except for the noted substitutions:

(SEQ ID NO: 189) 5′-TCGTCGAA*CGT*TCGAGATGAT (A* = 2-amino-dA; T* =2-thio-dT); (SEQ ID NO: 190) 5′-TCGTCGA*A*CGT*T*CGAGATGAT (A* =2-amino-dA; T* = 2-thio-dT); (SEQ ID NO: 187)5′-TCG*TCG*AACG*TTCG*AG*ATG*AT (G* = 7-deaza-8- aza-dG);(SEQ ID NO: 194) 5′-TCG*AACG*TTCG*AACG*TTCG*AACG*TT (G* = 7-deaza-8-aza-dG); (SEQ ID NO: 188) 5′-TCGTCGA*A*CGTTCGA*GA*TGA*T (A* =2-amino-dA); (SEQ ID NO: 197) 5′-TCGA*A*CGTTCGA*A*CGTTCGA*A*CGTT (A* =2-amino- dA).

PBMCs from eight donors were variously stimulated with either 0.2, 0.4,0.8, 1.6, 4.0 or 8 μg/ml of IMPs or controls and the resultantproduction of IFN-α was assessed as described above. The duplexes werecompared to the single sequences using the same total dose ofpolynucleotide (e.g., 4 μg/ml of SEQ ID NO:27 was compared to 4 μg/ml ofdouble strand SEQ ID NO:182 which contained 2 μg/ml SEQ ID NO:27 and 2μg/ml SEQ ID NO:29). The averaged result for the 8 donors at each IMPconcentration are reported in Table 12.

TABLE 12 Human PBMC Assays - IFN-α (pg/ml) IFN-α (pg/ml) test (SEQ ID 84 1.6 0.8 0.4 0.2 NO.) or control μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml 2(non-IMP) nd 87 nd nd nd nd  90 nd 77 nd nd nd nd 1 (IMP std) nd 288 nd77 77 nd 1 duplex 81 126 1988 1740 258  77  27 nd 8850 nd 955 77 nd  29nd 6040 nd 85 77 nd 182 (27/29 747 2162 6462 7280 1862  89 duplex) 187nd 1050 nd 139 77 nd 183 (187/29 91 117  311 1081 411 119 duplex) 188 nd644 nd 3360 147  nd 184 (188/29 225 978 5483 10057 5022 527 duplex) 189nd 845 nd 302 79 nd 185 (189/29 257 638 7345 7973 2711 314 duplex) 190nd 3064 nd 150 77 nd 186 (190/29 491 2673 6085 6603 1703 194 duplex) 194nd 164 nd 645 77 nd 197 nd 4833 nd 5742 1224 nd SAC (1:5000) 96 media 77nd = not determined

As can been seen in Table 12, the use of certain modified bases in theIMP can result in polynucleotides which have IFN-α stimulatory activity.With the exception of 183, these results also show that formation of aduplex polynucleotide with the complement sequence leads to a highlyactive IMP for stimulation of IFN-α production, particularly at lowerdoses. Polynucleotides which could not form duplexes on their own, e.g.,SEQ ID NO:189 and SEQ ID NO:190, induced little IFN-α while longersequences (e.g., SEQ ID NO:172, a 30-mer with a 28 base palindrome) andthe duplex SEQ ID NO:182 induced more IFN-α at low doses (e.g., 0.4 and0.8 μg/ml) than SEQ ID NO:27 and other IMPs with palindromes less than28 bases in length (as shown in Table 12 and FIG. 1). As discussedherein, certain modified bases can increase the stability of duplexesformed.

Example 2 Activation of Human B Cells by ImmunomodulatoryPolynucleotides

The ability of IMPs to activate human B cells was determined bymeasuring B cell proliferation and IL-6 production in response toincubation with IMPs. Human PBMCs were incubated with CD19 MACS beads(Miltenyi Biotec) and passed through a magnet, separating the CD19⁺ Bcells through positive selection (>98% CD19⁺ as determined by FACS). Forthe proliferation assay, B cells were cultured at 1×10⁵/well (5×10⁵/ml)in 96 well round-bottomed plates. Cells were incubated in triplicatewith 2 μg/ml IMP or control for 72 hours. At the end of the cultureperiod, the plates were pulsed with ³H-thymidine (1 μCi/well, Amersham)and incubated for an additional 8 hours. The plates were then harvestedand radioactive incorporation determined using standard liquidscintillation techniques, and the data was collected in counts perminute (cpm). For IL-6 secretion, B cells were cultured at0.5−1×10⁶/well in 48-well plates with 5 μg/ml IMP or control for 48hours, then culture supernatants were harvested and assayed for IL-6using ELISA with CytoSet antibody pairs according to manufacturer'sinstructions (BioSource). Limits of maximal/minimal detection were4000/2 pg/ml.

The results of the B cell proliferation assay presented in Table 13 arethe mean of the triplicate cell proliferation cpm values for cells fromeach donor and the mean of the cpm values for both donors. The resultsof the B cell IL-6 assay presented in Table 13 are the amount of IL-6produced from cells of each donor and the mean value from both donors.

TABLE 13 Human B Cell Assays test (SEQ ID Proliferation assay (cpm) IL-6assay (pg/ml) NO.) or control Donor 1 Donor 2 Mean Donor 1 Donor 2 Meanmedium 415 575 495 26 28 27 1 (IMP std) 27,731 43,403 35,567 222 531 3772 (non-IMP) 6748 7704 7226 52 126 89 43 22,695 26,456 24,576 187 935 56138 45,364 27,327 36,346 248 984 616 40 60,250 52,916 56,583 172 336 25419 22,683 29,569 26,126 173 257 215 LPS 1647 544 1096 34 21 28

From the results presented in Table 13, the compounds containing CGdinucleotides induced B cell proliferation and IL-6 production. As canbeen seen from the results presented in Table 9, although good B cellstimulatory activity in immunostimulatory polynucleotides is dependenton the presence of a CG dinucleotide, it does not appear to require themore specialized motifs described herein for high IFN-α induction.

In another assay, duplex forms of polynucleotides were compared tonon-duplex forms in the activation of B cells. Among the polynucleotidestested were: SEQ ID NO:1, SEQ ID NO:90, SEQ ID NO:27, and SEQ IDNO:182-duplex of SEQ ID NO:27 and SEQ ID NO:29. B cells from threedonors were stimulated with either 1.0 or 5.0 μg/ml of IMP or controland the resultant cell proliferation and IL-6 production was assessed asdescribed above. The averaged result for the 3 donors at each IMPconcentration is reported in Table 14.

TABLE 14 Human B Cell Assays Proliferation IL-6 assay test (SEQ ID NO.)assay (cpm) (pg/ml) or control 5 μg/ml 1 μg/ml 5 μg/ml 1 μg/ml  1 5792111307 554 73 27 66735 24529 723 322 182 (27/29 dpx) 78047 25344 809 28190 3333 2181 5 4 medium 2104 2104 4 4

From the results presented in Table 14, SEQ ID NO:182, the duplex formof SEQ ID NO:27 is approximately equivalent to SEQ ID NO:27 inactivating B cells as measured by stimulating IL-6 production and cellproliferation.

Example 3 Immunomodulation of Murine Cells by ImmunomodulatoryPolynucleotides

Immunomodulatory polynucleotides or control polynucleotides were assayedfor immunomodulatory activity on mouse splenocytes. The polynucleotidestested were fully modified phosphorothioate oligodeoxynucleotides. Amongthe polynucleotides tested were SEQ ID NO:1 (positive control) and SEQID NO:2 (negative control).

Fragments of BALB/c mouse spleen were digested with collagenase/dispase(0.1 U/mL/0.8 U/mL) dissolved in phosphate buffered saline (PBS) for 45minutes at 37° C., then mechanically dispersed by forcing the digestedfragments through metal screens. The dispersed splenocytes were pelletedby centrifugation, then resuspended in fresh medium (RPMI 1640 with 10%fetal calf serum, plus 50 units/mL penicillin, 50 μg/mL streptomycin, 2mM glutamine, and 0.05 mM β-mercaptoethanol).

Mouse splenocytes were dispensed into wells of 96 well plates (7×10⁷cells/ml) and incubated for one hour at 37° C. 100 μL of 2×concentration test sample or control was added and the cells wereincubated a further 24 hours. Each test sample or control was tested induplicate. Medium was harvested from each well and frozen at −80° C.before testing. Harvested medium was thawed and tested for cytokineconcentrations by ELISA. Polynucleotides were tested at variousconcentrations including 5.0, 1.0 and 0.1 μg/ml. Among thepolynucleotides tested were 5′-TGACTGTGAACGTTCGAAATGA (SEQ ID NO:36) and5′-TGACTGTGAACGTTCGAAGTGA (SEQ ID NO:37). Control samples included mediaalone and PANSORBIN® heat-killed, formalin-fixed Staphylococcus aureus(SAC) (CalBiochem).

IL-6, IL-12 and IFN-γ was assayed using a sandwich-format ELISA. Mediumfrom the mouse splenocyte assay was incubated in microtiter platescoated with anti-IL-6, anti-IL-12 p40/p70 or anti-IFN-γ monoclonalantibody (Nunc). Bound cytokine (IL-6, IL-12 or IFN-γ) was detectedusing a biotinylated anti-cytokine antibody (anti-IL-6, anti-IL-12p40/p70 or anti-IFN-γ) and streptavidin-horseradish peroxidaseconjugated secondary antibody, developed with the chromogenic peroxidasesubstrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence ofperoxidase, and quantitated by measuring absorbance at 450 nm using aEmax precision microplate reader (Molecular Devices). Values of IL-6less than 45 pg/ml were assigned a value of 45 pg/ml (i.e., 45=<45).Values of IL-12 p40/p70 less than 36 pg/ml were assigned a value of 36pg/ml (i.e., 36=<36). Values of IFN-γ less than 54 pg/ml were assigned avalue of 54 pg/ml (i.e., 54=<54).

Tables 15 and 16 summarize assay results for cytokine production inresponse to IMPs. Immunomodulatory polynucleotides containing a CGdinucleotide generally stimulated IL-6, IL-12 and IFN-γ secretion bymurine splenocytes irrespective of the presence of the more specializedmotifs described herein for high IFN-α induction.

TABLE 15 Murine Splenocyte Assay - IL-6 (pg/ml) Test (SEQ ID NO.) orControl Dose (ug/ml) Rep. 1 Rep. 2 Ave. 1 (IMP std) 5.0 4623 4655 46391.0 999 961 980 0.1 47 45 46 2 (non-IMP) 5.0 45 45 45 1.0 45 45 45 SAC308 296 302 Media — — 45  5 5.0 4755 4653 4704 1.0 1055 985 1020 0.1 4546 46 20 5.0 4953 5464 5209 1.0 1318 1413 1366 0.1 90 124 107 19 5.04421 4726 4574 1.0 645 740 693 0.1 45 45 45 38 5.0 4267 4350 4309 1.0613 673 643 0.1 89 160 125 39 5.0 4775 4819 4797 1.0 802 731 767 0.1 213147 180 40 5.0 2644 2217 2431 1.0 341 251 296 0.1 45 45 45 43 5.0 101105 103 1.0 45 45 45 0.1 45 45 45 27 5.0 4809 5245 5027 1.0 2182 26932438 0.1 216 242 229 41 5.0 4781 5504 5143 1.0 1979 2285 2132 0.1 316372 344 42 5.0 2706 3242 2974 1.0 460 577 519 0.1 66 70 68 44 5.0 24582585 2522 1.0 358 321 340 0.1 45 45 45 45 5.0 3920 3667 3794 1.0 11771117 1147 0.1 45 45 45 46 5.0 45 45 45 1.01 45 45 45 0.1 45 45 45 47 5.0163 213 188 1.0 45 45 45 0.1 45 45 45 48 5.0 182 216 199 1.0 45 45 450.1 45 45 45 49 5.0 690 765 728 1.0 66 73 70 0.1 45 45 45 50 5.0 45 4545 1.0 45 45 45 0.1 45 45 45 51 5.0 1942 1868 1905 1.0 224 197 211 0.145 45 45 52 5.0 1421 1234 1328 1.0 456 488 472 0.1 45 45 45 36 5.0 36563834 3745 1.0 858 991 925 0.1 45 45 45 36 5.0 3716 3750 3733 1.0 897 934916 0.1 45 45 45 37 5.0 4253 4643 4448 1.0 1256 1218 1237 0.1 157 190174 37 5.0 4457 4323 4390 1.0 1099 941 1020 0.1 88 109 99

TABLE 16 Murine Splenocyte Assay - IL-12 & IFN-γ Dose IL-12 (pg/ml)IFN-γ (pg/ml) Test (SEQ ID (ug/ Rep. Rep. Rep. Rep. NO) or Control ml) 12 Ave. 1 2 Ave. 1 (IMP std) 5.0 1915 1737 1826 1858 2589 2089 1.0 14191424 1422 1941 1954 1948 0.1 573 603 588 179 395 287 2 (non-IMP) 5.0 3836 37 54 54 54 1.0 36 43 40 54 54 54 SAC 609 620 615 11889 13338 12614media — — 44 — — 54  5 5.0 1773 1679 1726 1331 1463 1397 1.0 2099 21932146 1878 1811 1845 0.1 651 649 650 271 157 214 20 5.0 1838 2023 19312822 3342 3082 1.0 2245 2315 2280 2662 3402 3032 0.1 1016 1077 1047 5131392 953 19 5.0 1364 1458 1411 1997 2686 2343 1.0 1513 1702 1608 14272375 1901 0.1 648 597 623 58 54 56 38 5.0 1822 1870 1846 3168 3851 35101.0 1963 2239 2101 3440 3721 3581 0.1 1207 1430 1319 446 1364 905 39 5.02476 2344 2410 3578 3065 3322 1.0 2856 2504 2680 2415 3497 2956 0.1 21012085 2093 1403 1217 1310 40 5.0 902 797 850 605 502 554 1.0 1244 12161230 1116 318 717 0.1 304 210 257 54 54 54 43 5.0 940 720 830 54 54 541.0 721 852 787 54 54 54 0.1 37 36 37 54 54 54 27 5.0 1978 2295 21373603 4546 4075 1.0 1833 2373 2103 3634 4735 4185 0.1 1761 1945 1853 24012313 2357 41 5.0 1590 1898 1744 3328 4447 3888 1.0 1611 1910 1761 41973402 3800 0.1 1738 1853 1796 3030 3016 3023 42 5.0 1507 1887 1697 27473203 2975 1.0 2185 2269 2227 2609 4162 3386 0.1 669 669 669 192 206 19944 5.0 1870 1805 1838 2593 2802 2698 1.0 2058 1854 1956 1464 1747 16060.1 235 214 225 54 54 54 45 5.0 1716 1597 1657 2153 1776 1965 1.0 13411175 1258 1567 1368 1468 0.1 646 446 546 54 54 54 46 5.0 525 392 459 5454 54 1.0 234 132 183 54 54 54 0.1 36 36 36 54 54 54 47 5.0 746 738 74254 54 54 1.0 757 752 755 54 54 54 0.1 59 64 62 54 54 54 48 5.0 578 676627 54 54 54 1.0 697 786 742 54 54 54 0.1 41 51 46 54 54 54 49 5.0 10951288 1192 376 778 577 1.0 1510 1551 1531 54 54 54 0.1 79 111 95 54 54 5450 5.0 586 424 505 54 54 54 1.0 206 178 192 54 54 54 0.1 39 44 42 54 5454 51 5.0 1341 1117 1229 955 1023 989 1.0 1412 1257 1335 426 845 636 0.192 75 84 54 54 54 52 5.0 1855 1557 1706 2408 2107 2258 1.0 2961 2821 1984421 5632 5027 0.1 205 245 225 54 934 494 36 5.0 1717 1656 1687 33903338 3364 1.0 1480 1510 1495 2547 2832 2690 0.1 700 571 636 384 264 32436 5.0 1478 1565 1522 2281 2200 2241 1.0 1293 1235 1264 2073 3112 25930.1 666 590 628 54 448 251 37 5.0 1679 1918 1799 3240 3748 3494 1.0 16031561 1582 3950 4437 4194 0.1 1232 1235 1234 1548 2044 1796 37 5.0 20643202 2633 2419 2631 2525 1.0 1895 2417 2156 1894 3332 2613 0.1 831 14301131 293 530 412

From the results presented in Tables 15 and 16, all compounds containingCpG motifs induced IL-12 production from murine splenocytes and most,but not all, compounds containing CpG motifs induced IL-6 and IFN-γproduction from murine splenocytes. As can been seen from the resultspresented in Tables 15 and 16, although IL-6, IL-12 and IFN-γstimulatory activity of immunostimulatory polynucleotides on murinesplenocytes is generally dependent on the presence of a CG dinucleotide,it does not appear to require the more specialized motifs describedherein for high IFN-α induction.

Example 4 Stimulation of Interferon-Inducible Gene Expression byImmunomodulatory Polynucleotides

As demonstrated herein, immunomodulatory polynucleotides can induceproduction of IFN-γ and/or IFN-α from PBMCs. IMPs were assayed foractivity on human PBMCs for inducing mRNA expression of additionalcytokine genes, chemokine genes and other genes using a quantitative PCRtechnique, the TaqMan technique. The polynucleotides tested were fullymodified phosphorothioate oligodeoxynucleotides. Among thepolynucleotides tested were SEQ ID NO:1 (positive control) and SEQ IDNO:2 (negative control).

Human PMBCs were prepared as described in Example 1. The cells werecultured in the presence of test samples (IMPs or controls) at 5 μg/mlμg/ml for 24 hours. Total RNA was extracted using the Qiagen RNeasy MiniProtocol (Qiagen) and converted to cDNA using oligo dT (Promega), randomhexamers (Promega), and SuperScript RT II (InVitrogen). cDNA was diluted1:10 and PCR conducted using either QuantiTect SYBR green PCR master mix(Qiagen) and naked primers (synthesized by Operon) or QuantiTect probePCR master mix (Qiagen) and PDAR primers with labeled probe (AppliedBioSystems). Reactions were conducted using the GeneAmp 5700 SequenceDetector (PE BioSystems).

Examples of the sequences for synthesized primers are as follows (listed5′ to 3′):

Ubiquitin (F: CACTTGGTCCTGCGCTTGA (SEQ ID NO: 200),R: CAATTGGGAATGCAACAACTTTAT (SEQ ID NO: 201)); 2,5-OAS(F: AGGGAGCATGAAAACACATTTCA (SEQ ID NO: 202),R: TTGCTGGTAGTTTATGACTAATTCCAAG (SEQ ID NO: 203)); GBP-1(F: TGGAACGTGTGAAAGCTGAGTCT (SEQ ID NO: 204),R: CATCTGCTCATTCTTTCTTTGCA (SEQ ID NO: 205)); IFN-α(F: CCCAGGAGGAGTTTGGCAA (SEQ ID NO: 206),R: TGCTGGATCATCTCATGGAGG (SEQ ID NO: 207)); ISG-54K(F: CTGGACTGGCAATAGCAAGCT (SEQ ID NO: 208),R: AGAGGGTCAATGGCGTTCTG (SEQ ID NO: 209)); MCP-2(F: CTGCTCATGGCAGCCACTTT (SEQ ID NO: 210),R: AGCAGGTGATTGGAATGGAAA (SEQ ID NO: 211)); MIG(F: CATCTTGCTGGTTCTGATTGGA (SEQ ID NO: 212),R: TGGTGCTGATGCAGGAACAG (SEQ ID NO: 213)); TNF-α(F: CTTCTGCCTGCTGCACTTTG (SEQ ID NO: 214),R: CTGGGCCAGAGGGCTGAT (SEQ ID NO: 215)).

IFN-γ, IL-1α, IL-6, IP-10, MCP-3, and MIP-3β, were measured using PDARssupplied by PE BioSystems. Threshold cycle (C_(T)) values for each genewere normalized to ubiquitin using the formula1.8^((UBQ-GENE))(100,000), where UBQ is the mean C_(T) of triplicateubiquitin runs, GENE is the mean C_(T) of duplicate runs of the gene ofinterest, and 100,000 is arbitrarily chosen as a factor to bring allvalues above 0. The negative control for each experiment, stimulationwith medium alone, is assigned a value of 1 and all data is expressed asfold induction over the negative control.

Table 17 summarizes assay results for cytokine, chemokine andinflammatory protein gene expression from PBMCs in response to the IMPSEQ ID NO:27. Also tested was polynucleotide 5′-GGTGCATCGATGCAGGGGGG(SEQ ID NO:154). Data is presented as the mean of fold induction overmedium control (given the value of 1.0) with SEM.

TABLE 17 Profile of gene expression modulated by IMP Test or Control(SEQ ID IL-1α IP-10 MCP-2 MCP-3 MIG MIP-3β 2,5-OAS GBP-1 ISG-54K NO)mean SEM mean SEM mean SEM mean SEM mean SEM mean SEM mean SEM mean SEMmean SEM medium 1.0 0.0 1.0 0.0 1.0 0.0 1.0 0.0 1.0 0.0 1.0 0.0 1.0 0.01.0 0.0 1.0 0.0 2 2.0 0.7 0.6 0.3 0.2 0.1 0.9 0.1 0.6 0.1 1.2 0.3 0.70.2 1.0 0.1 0.7 0.1 1 1.7 0.4 2.7 0.6 28.3 21.2 3.0 1.0 3.0 0.9 2.9 0.97.6 3.3 2.5 0.6 4.9 2.1 27  0.4 0.2 94.0 27.5 198.8 59.6 8.0 2.2 8.8 2.06.9 1.8 16.5 2.3 5.9 0.4 27.1 2.6 154  0.2 0.1 145.4 65.1 284.8 108.78.5 1.4 14.5 7.0 10.5 2.1 15.7 1.3 5.7 1.1 31.9 2.1

As shown in Table 17, SEQ ID NO:27 strongly increased expression of thechemokines IP-10, MCP-2, MCP-3, MIG, and MIP-3β. The expression of IL-1αdecreased in the presence of SEQ ID NO:27. In addition, SEQ ID NO:27markedly increased expression of the IFN-α-inducible genes2,5-oligoadenylate synthetase (2,5-OAS), interferon-stimulating gene-54K(ISG-54K), and guanylate-binding protein-1 (GBP-1).

In these assays, the IMP SEQ ID NO:27 had no significant effect on theexpressed mRNA levels of the cytokines G-CSF, IL-1β, IL-6, IL-12 p40,IL-23, TNF-α, or of the chemokines BCA-1, IL-8, LPTN, MCP-1, MDC,MIP-1a, MIP-1b, MIP-3a, RANTES, and TARC.

Example 5 Stimulation of NK Cell Lytic Activity by ImmunomodulatoryPolynucleotides

IMPs of the present invention stimulate improved natural killer (NK)cell lytic activity as compared to an IMP standard. NK cell lyticactivity was assayed through lysis of K562 target cells. In brief, PBMCswere stimulated with 10 mg/ml IMP (previously obtained optimal dose) ornegative control polynucleotide for 48 hours in culture. The treatedPBMCs were then co-cultured with ⁵¹Cr-loaded K562 tumor target cells ata range of effector:target ratios for 4 hours. ⁵¹Cr released upon celllysis was measured by a TopCount NXT scintillation counter (Packard) andreported as counts per minute (cpm).

Results of NK cell stimulation from two different PBMC donors is shownin FIG. 2. The IMPs used in the assays were SEQ ID NO:1, SEQ ID NO:90,SEQ ID NO:27, SEQ ID NO:172 and SEQ ID NO:113. The palindrome length inthe IMPs is: 28 bases in SEQ ID NO:172, 22 bases in SEQ ID NO:113, 12bases in SEQ ID NO:27, and 8 bases in SEQ ID NO:1. SEQ ID NO:90, anon-IMP control, has a palindrome length of 20 bases but does notcontain a 5′-C, G-3′ sequence. In this experiment, IMPs with palindromesof 12 bases in length or longer stimulated an increased amount NK celllytic activity as compared to the IMP standard SEQ ID NO:1 with apalindrome length of 8 bases.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore,descriptions and examples should not be construed as limiting the scopeof the invention.

1. An immunomodulatory polynucleotide, comprising: a)5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGX₂′X₁′(CG)_(p))_(z) (SEQ ID NO: 156)wherein N are nucleosides, x=0, y=1, w=0, p=0 or 1, q=0, 1 or 2, andz=1-20, X₁ and X₁′ are self-complimentary nucleosides, X₂ and X₂′ areself-complimentary nucleosides, and wherein the 5′ T of the(TCG(N_(q)))_(y) sequence is positioned at the 5′ end of thepolynucleotide; and b) a palindromic sequence at least 8 bases in lengthwherein the palindromic sequence comprises the first (X₁X₂CGX₂′X₁′) ofthe (X₁X₂CGX₂′X₁′(CG)_(p))_(z) sequences, wherein the polynucleotide isat least 15 bases in length.
 2. An immunomodulatory polynucleotideaccording to claim 1 wherein the palindromic sequence has a basecomposition of more than one-third A's and T's.
 3. An immunomodulatorypolynucleotide according to claim 1 wherein the wherein the IMPcomprises a sequence selected from the group consisting of SEQ ID NO:172, SEQ ID NO: 55, SEQ ID NO: 113, SEQ ID NO: 39, SEQ ID NO: 53, SEQ IDNO: 109, SEQ ID NO: 117, SEQ ID NO: 175, SEQ ID NO: 147, SEQ ID NO: 148,SEQ ID NO: 80, SEQ ID NO: 97, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:152, SEQ ID NO: 153, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQID NO: 173, SEQ ID NO: 174, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83,SEQ ID NO: 84, SEQ ID NO: 79, SEQ ID NO: 77, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 43, SEQ IDNO: 78, SEQ ID NO: 118, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178,SEQ ID NO: 179, and SEQ ID NO:
 180. 4. An immunomodulatory compositioncomprising an immunomodulatory polynucleotide according to claim 1 or 3.5. A method of modulating an immune response in an individualcomprising: administering to an individual an immunomodulatorypolynucleotide according to claim 1 or 3 in an amount sufficient tomodulate an immune response in said individual.
 6. A method ofincreasing interferon-gamma (IFN-γ) in an individual, comprising:administering an immunomodulatory polynucleotide according to accordingto claim 1 or 3 to said individual in an amount sufficient to increaseIFN-γ in said individual.
 7. A method of increasing interferon-alpha(IFN-α) in an individual, comprising: administering an immunomodulatorypolynucleotide according to claim 1 or 3 to said individual in an amountsufficient to increase IFN-α in said individual.
 8. A method ofameliorating a symptom of an infectious disease in an individual,comprising: administering an effective amount of an immunomodulatorypolynucleotide according to claim 1 or 3 to the individual, wherein aneffective amount is an amount sufficient to ameliorate a symptom of saidinfectious disease.
 9. A method of ameliorating a symptom of anIgE-related disorder in an individual, comprising: administering aneffective amount of an immunomodulatory polynucleotide according toclaim 1 or 3 to an individual having an IgE-related disorder, wherein aneffective amount is an amount sufficient to ameliorate a symptom of saidIgE-related disorder.
 10. An immunomodulatory composition comprising animmunomodulatory polynucleotide according to claim 1 or 3 wherein theimmunomodulatory polynucleotide comprises a modification of one or morephosphate groups.
 11. The immunomodulatory composition of claim 10wherein the modification of one or more phosphate groups is aphosphorothioate linkage.
 12. The immunomodulatory composition of claim10 wherein the modification of one or more phosphate groups arephosphorothioate and phosphodiester linkages.